Insulin and IGF-1 Receptor Agonists and Antagonists

ABSTRACT

Peptide sequences capable of binding to insulin and/or insulin-like growth factor receptors with either agonist or antagonist activity and identified from various peptide libraries are disclosed. This invention also identifies at least two different binding sites, which are present on insulin and insulin-like growth factor receptors, and which selectively bind the peptides of this invention. As agonists, the peptides of this invention may be useful for development as therapeutics to supplement or replace endogenous peptide hormones. The antagonist peptides may also be developed as therapeutics.

I. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/410,222 filed Apr. 24, 2006 which is a continuation of U.S.application Ser. No. 10/253,493 filed Sep. 24, 2002 which is acontinuation-in-part of U.S. application Ser. No. 09/962,756 filed Sep.24, 2001, which is a continuation-in-part of U.S. application Ser. No.09/538,038 filed Mar. 29, 2000, which is a continuation-in-part of U.S.application Ser. No. 09/146,127, filed Sep. 2, 1998, all of which areincorporated herein by reference in their entirety.

II. FIELD OF THE INVENTION

This invention relates to the field of hormone receptor activation orinhibition. More specifically, this invention relates to theidentification of molecular structures, especially peptides, which arecapable of acting at either the insulin or insulin-like growth factorreceptors as agonists or antagonists. Also related to this invention isthe field of molecular modeling whereby useful molecular models arederived from known structures.

III. SEQUENCE LISTING

The parent application of U.S. application Ser. No. 11/410,222 filedApr. 24, 2006, U.S. application Ser. No. 10/253,493 filed Sep. 24, 2002,contains a “lengthy” sequence listing which has been submitted via CD-Rin lieu of a printed paper copy, and is hereby incorporated by referencein its entirety. Said CD-R, recorded on Apr. 21, 2006, are labeled“CRF,” “Copy 1” and “Copy 2”, respectively, and each contains only oneidentical 932 Kb file (65975US2.APP).

IV. BACKGROUND OF THE INVENTION

Insulin is a potent metabolic and growth promoting hormone that acts oncells to stimulate glucose, protein, and lipid metabolism, as well asRNA and DNA synthesis. A well-known effect of insulin is the regulationof glucose levels in the body. This effect occurs predominantly inliver, fat, and muscle tissue. In the liver, insulin stimulates glucoseincorporation into glycogen and inhibits the production of glucose. Inmuscle and fat tissue, insulin stimulates glucose uptake, storage, andmetabolism. Defects in glucose utilization are very common in thepopulation, giving rise to diabetes.

Insulin initiates signal transduction in target cells by binding to aspecific cell-surface receptor, the insulin receptor (IR). The bindingleads to conformational changes in the extracellular domain of IR, whichare transmitted across the cell membrane and result in activation of thereceptor's tyrosine kinase activity. This, in turn, leads toautophosphorylation of tyrosine kinase of IR, and the binding of solubleeffector molecules that contain SH2 domains such asphophoinositol-3-kinase, Ras GTPase-activating protein, andphospholipase Cγ to IR (Lee and Pilch, 1994, Am. J. Physiol.266:C319-C334).

Insulin-like growth factor 1 (IGF-1) is a small, single-chain protein(MW=7,500 Da) that is involved in many aspects of tissue growth andrepair. It is similar in size, sequence, and structure to insulin, buthas 100-1,000-fold lower affinity for IR (Mynarcik et al., 1997, J.Biol. Chem. 272:18650-18655). Although IGF-1 mRNA can be detected inmany tissues, the majority of circulating IGF-1 is produced in the liverafter stimulation by growth hormone (Butt et al., 1999, Immunol. CellBiol. 77:256-262). Functionally, IGF-1 appears to act as a mitogen andas an anti-apoptotic factor for cells.

Recent studies have analyzed the role of endogenous IGF-1 in variousdisease states. Several reports have shown that IGF-1 promotes thegrowth of normal and cancerous prostate cells both in vitro and in vivo(Angelloz-Nicoud and Binoux, 1995, Endocrinol. 136:5485-5492; Figueroaet al., 1995, J. Clin. Endocrinol. Metab. 80:3476-3482; Torring et al.,1997, J. Urol. 158:222-227). Elevated serum levels of IGF-1 have beenshown to be associated with increased risks of prostate cancer, and maybe an earlier predictor of onset than prostate-specific antigen (PSA; J.M. Chan et al., 1998, Science 279:563-566). Serum levels of free IGF-1are regulated by the presence of IGF binding proteins (IGFBP), whichbind to IGF-1 and prevent its interaction with the IGF-1R (reviewed inC. A. Conover, 1996, Endocr. J. 43S:S43-S48; Rajaram et al., 1997,Endocr. Rev. 18:801-831). PSA has been shown to be a protease thatcleaves IGFBP-3, resulting in an increase of free IGF-1 in serum (P.Cohen et al., 1992, J. Clin. Endocrinol. Metab. 75:1046-1053; P. Cohenet al., 1994, J. Endocrinol. 142:407-415; H. Lilja, 1995, Scand. J.Clin. Lab. Invest. Suppl. 220:47-56). Consistent with this finding, menwith higher levels of circulating IGF-1 and lower levels of IGFBP-3 werefound to be at higher risk for developing colorectal cancer (J. Ma etal., 1999, J. Natl. Cancer Instit. 91:620-625.). Recent studies havealso shown a connection between IGF-1 levels and ovarian cancer.

There also appears to be a relationship between high levels of IGF-1and/or IGF-1R and breast cancer (L. C. Happerfield et al., 1997, J.Pathol. 183:412-417). A positive correlation was observed betweencirculating IGF-1 and breast cancer among pre-menopausal women (S. E.Hankinson et al., 1998, Lancet 351:1393-1396). A poor prognosis forbreast cancer patients was correlated to the expression of IGF-1Rpositive and estrogen receptor (ER) negative cells (A. A. Butler et al.,1998, Cancer Res. 58:3021-3027). Recently, investigators have identifiedhybrid IGF-1R/IR receptors found in several breast cancer cell lines (G.Pandini et al., 1999, Clin. Cancer Res. 5:1935-1944; E. M. Bailyes etal., 1997, Biochem. J. 327(Pt 1):209-215; see below). The data hassuggested that these hybrids behave as functional IGF-1Rs and may play amajor role in IGF-1 signaling in breast cancer.

Clinical studies have also investigated the use of recombinant humanIGF-1 in the treatment of several diseases, including type I diabetes(Carroll et al., 1997, Diabetes 46:1453-1458; Crowne et al., 1998,Metabolism 47:31-38), amyotropic lateral sclerosis (Lai et al., 1997,Neurology 49:1621-1630), and diabetic motor neuropathy (Apfel andKessler, 1996, CIBA Found. Symp. 196:98-108). Other potentialtherapeutic applications of IGF-1, such as osteoporosis (Canalis, 1997,Bone 21:215-216), immune modulation (Clark, 1997, Endocr. Rev.18:157-179) and nephrotic syndrome (Feld and Hirshberg, 1996, Pediatr.Nephrol. 10:355-358) are also under investigation. Clearly, IGF-1Ractivity is involved in many disease states, indicating that there arepotential clinical applications for both IGF-1 agonists and antagonists.

Both insulin and IGF-1 are expressed as precursor proteins comprising,among other regions, contiguous A, B, and C peptide regions, with the Cpeptide being an intervening peptide connecting the A and B peptides. Amature insulin molecule is composed of the A and B chains connected bydisulfide bonds, where the connecting C peptide has been removed duringpost-translational processing. IGF-1 retains its smaller C-peptide aswell as a small D extension at the C-terminal end of the A chain, makingthe mature IGF-1 slightly larger than insulin (Blakesley, 1996). The Cregion of human IGF-1 appears to be required for high affinity bindingto IGF-1R (Pietrzkowski et al., 1992, Cancer Res. 52(23):6447-51).Specifically, tyrosine 31 located within this region appears to beessential for high affinity binding. Furthermore, deletion of the Ddomain of IGF-1 increased the affinity of the mutant IGF-1 for bindingto the IR, while decreasing its affinity for the IGF-1R (Pietrzkowski etal., 1992). A further distinction between the two hormones is that,unlike insulin, IGF-1 has very weak self-association and does nothexamerize (De Meyts, 1994).

IGF-1 and insulin competitively cross-react with IGF-1R and IR (L.Schäffer, 1994, Eur. J. Biochem. 221:1127-1132). Yet, despite 45%overall amino acid identity, insulin and IGF-1 bind only weakly to eachother's receptor. The affinity of each peptide for the non-cognatereceptor is about 3 orders of magnitude lower than that for the cognatereceptor (Mynarcik, et al., 1997, J. Biol. Chem. 272:18650-18655). Thedifferences in binding affinities may be partly explained by thedifferences in amino acids and unique domains which contribute to uniquetertiary structures of ligands (Blakesley et al., 1996, Cytokine GrowthFactor Rev. 7(2):153-9).

IGF-1R and IR are related members of the tyrosine-kinase receptorsuperfamily of growth factor receptors. Another family member isinsulin-related receptor (IRR), for which no natural ligand is known.Both IGF-1R and IR are comprised of two α and two β subunits which forma disulfide-linked heterotetramer (β-α-α-β). These receptors have anextracellular ligand binding domain, a single transmembrane domain, anda cytoplasmic domain displaying the tyrosine kinase activity. Theextracellular domain is composed of the entire α subunits and a portionof the N-terminus of the β subunits, while the intracellular portion ofthe β subunits contains the tyrosine kinase domain. In contrast to othertyrosine kinase receptors, IGF-1R, IR and IRR exist on the cell surfaceas disulphide-linked dimers and require domain rearrangements ratherthan receptor oligomerization for cell signaling (Adams et al., 2000,Cell. Mol. Life. Sci. 57:1050-1093; Garrett et al., 1998, Nature394:395-399; Frasca et al., 1999, Mol. Cell. Biol. 19: 3278-3288; DeMeyts et al., 1994, Hormone Res. 42:152-169). In addition, insulin andIGF-1 hemireceptors (comprising one α subunit and one β subunit) canheterodimerize to form IR/IGF-1R hybrids (M. A. Soos et al., 1990,Biochem. J. 270:383-390; J. Kasua et al., 1993, Biochemistry32:13531-13536; B. L. Seely et al., 1995, Endocrinology 136:1635-1641).

In many cells, IR/IGF-1R hybrids are the most common receptor subtype(Bailyes et al., 1997, Biochem. J. 327(pt. 1):209-215). The proportionof total IGF-1R assembled into hybrids varies between 40% and 60% inhuman tissues (M. Federici et al., 1997, Mol. Cell. Endocrin.129(2):121-6). IR/IGF-1R hybrids are also overproduced in human cancercells as a result of overexpression of IR and IGF-1R (Pandini et al.,1999, Clin. Cancer Res. 5:1935-1944; A. Belfiore et al., 1999,Biochemie, 81(4):403-7; V. Papa et al., 1990, J. Clin. Invest.86:1503-1510; V. Papa et al., 1993, Cancer Res. 53:3736-3740). Inparticular, increased levels of IR/IGF-1R hybrids have been observed inbreast cancer cell lines and breast cancer tissue specimens (Pandini etal., 1999, Clin. Cancer Res. 5:1935-1944). Similarly, high levels ofIR/IGF-1R hybrids have been observed in thyroid cancer specimens andcell lines (A. Belfiore et al., 1999, Biochemie, 81(4):403-7).Functional studies have indicated that IR/IGF-1R hybrids arepredominantly activated by IGF-1 (M. A. Soos et al., 1993, Biochem. J.290(pt. 2):419-426; A. L. Frattali et al., 1993, J. Biol. Chem.268:7393-7400). Accordingly, it has been postulated that IR/IGF-1Rhybrids provide additional binding sites for IGF-1, and thereby increasecell sensitivity to this factor (Bailyes et al., 1997, Biochem. J.327(pt. 1):209-215; Pandini et al., 1999, Clin. Cancer Res. 5:1935-1944;A. Belfiore et al., 1999, Biochemie, 81(4):403-7).

IR is a glycoprotein having molecular weight of 350-400 kDa (dependingof the level of glycosylation). It is synthesized as a singlepolypeptide chain and proteolytically cleaved to yield adisulfide-linked monomer α-β insulin receptor. Two α-β monomers arelinked by disulfide bonds between the α-subunits to form a dimeric formof the receptor (β-α-α-β-type configuration). The α subunit is comprisedof 723 amino acids, and it can be divided into two large homologousdomains, L1 (amino acids 1-155) and L2 (amino acids 313-468), separatedby a cysteine-rich region (amino acids 156-312) (Ward et al., 1995,Prot. Struct. Funct. Genet. 22:141-153). Many determinants of insulinbinding seem to reside in the α-subunit. The β-subunit of IR has 620amino acid residues and three domains: extracellular, transmembrane, andcytosolic. The extracellular domain is linked by disulfide bridges tothe α-subunit. The cytosolic domain includes the tyrosine kinase domain,the three-dimensional structure of which has been solved (Hubbard etal., 1994, Nature 372:746-754). A unique feature of IR is that it isdimeric in the absence of ligand.

To aid in drug discovery efforts, a soluble form of a membrane-boundreceptor was constructed by replacing the transmembrane domain and theintracellular domain of IR with constant domains from immunoglobulin Fcor λ subunits (Bass et al., 1996, J. Biol. Chem. 271:19367-19375). Therecombinant gene was expressed in human embryonic kidney 293 cells. Theexpressed protein was a fully processed heterotetramer and the abilityto bind insulin was similar to that of the full-length holoreceptor.

IGF-1R is synthesized as a 180 kDa precursor which is glycosylated,dimerized and proteolytically processed to yield mature receptor (T. E.Adams et al., 2000, Cell. Mol. Life Sci., 57:1050-1093, 2000). Themature receptor/complex consists of two extracellular α-subunits and twotransmembrane β-subunits having tyrosine kinase activity. IGF-1R isexpressed in almost all normal adult tissue except for liver, which isitself the major site of IGF-1 production (Butt et al., 1999, Immunol.Cell Biol. 77:256-262). A variety of signaling pathways are activatedfollowing binding of IGF-1 to the IGF-1R, including Src and ras, as wellas downstream pathways, such as the MAP kinase cascade and the PI3K/AKTaxis (Chow et al., 1998, J. Biol. Chem. 273:4672-4680).

The sequence of IR is highly homologous to the sequence of IGF-1R,indicating that the three-dimensional structures of both receptors maybe similar. The α-subunits, which contain the ligand binding region ofIR and IGF-1R, exhibit between 47-67% overall amino acid identity. Threegeneral domains, termed L1, cysteine-rich, and L2, have been reportedfor both receptors from sequence analysis of the α subunits. Thecysteine residues in the cysteine-rich region are highly conservedbetween the two receptors; however, the cysteine-rich regions share only48% overall amino acid identity. Notably, the crystal structure of thefirst three domains of IGF-1R has been determined (Garrett et al., 1998,Nature 394:395-399). The L domains consist of a single-strandedright-handed β-helix (a helical arrangement of β-strands), while thecysteine-rich region is composed of eight disulfide-bonded modules.

While similar in structure, IGF-1R and IR serve different physiologicalfunctions. IR is primarily involved in metabolic functions whereasIGF-1R mediates growth and differentiation. Consistent with this,ablation of IGF-1 (i.e., in IGF-1 knock-out mice) results in embryonicgrowth deficiency, impaired postnatal growth, and infertility. Inaddition, IGF-1R knock-out mice were only 45% of normal size and died ofrespiratory failure at birth (Liu et al., 1993, Cell 75:59-72). However,both insulin and IGF-1 can induce both mitogenic and metabolic effects.Whether each ligand elicits both activities via its own receptor, orwhether insulin exerts its mitogenic effects through its weak affinitybinding to IGF-1R, and IGF-1 its metabolic effects through IR, remainscontroversial (De Meyts, 1994, Horm. Res. 42:152-169).

Also, despite the similarities observed between these two receptors, therole of the domains in specific ligand binding are distinct. Throughchimeric receptor studies, (domain swapping of the IR and IGF-1Rα-subunits), researchers have reported that the sites of interaction ofthe ligands with their specific receptors differ (T. Kjeldsen et al.,1991, Proc. Natl. Acad. Sci. USA 88:4404-4408; A. S. Andersen et al.,1992, J. Biol. Chem. 267:13681-13686). For example, the cysteine-richdomain of the IGF-1R was determined to be essential for high-affinityIGF binding, but not insulin binding. When amino acids 191-290 of IGF-1Rregion was introduced into the corresponding region of the IR (aminoacids 198-300), the modified IR bound both IGF-1 and insulin with highaffinity. Conversely, when the corresponding region of the IR wasintroduced into the IGF-1R, the modified IGF-1R bound to IR but notIGF-1.

A further distinction between the binding regions of the IR and IGF-1Ris their differing dependence on the N-terminal and C-terminal regions.Both the N-terminal and C-terminal regions (located within the putativeL1 and L2 domains) of the IR are important for high-affinity insulinbinding but appear to have little effect on IGF-1 binding for either IRor IGF-1R. Replacing residues in the N-terminus of IGF-1R (amino acids1-62) with the corresponding residues of IR (amino acids 1-68) confersinsulin-binding ability on IGF-1R. Within this region, residues Phe-39,Arg-41 and Pro-42 are reported as major contributors to the interactionwith insulin (Williams et al., 1995). When these residues are introducedinto the equivalent site of IGF-1R, the affinity for insulin is markedlyincreased, whereas, substitution of these residues by alanine in IRresults in markedly decreased insulin affinity. Similarly, the regionbetween amino acids 704-717 of the C-terminus of IR has been shown toplay a major role in insulin specificity. Substitution of these residueswith alanine also disrupts insulin binding (Mynarcik et al., 1996, J.Biol. Chem. 271(5):2439-42; C. Kristensen et al., 1999, J. Biol. Chem.274(52):37351-37356).

Alanine scans of IR and IGF-1R suggest that insulin and IGF-1 may usesome common contacts to bind to IGF-1R but that those contacts differfrom those that insulin utilizes to bind to IR (Mynarcik et al., 1997).Hence, the data in the literature has led one commentator to state thateven though “the binding interfaces for insulin and IGF-1 on theirrespective receptors may be homologous within this interface the sidechains which make actual contact and determine specificity may be quitedifferent between the two ligand-receptor systems” (De Meyts, 1994).

Based on data for binding of insulin and insulin analogs to variousinsulin receptor constructs, a binding model has been proposed. Thismodel shows insulin receptor with two insulin binding sites that arepositioned on two different surfaces of the receptor molecule, such thateach alpha-subunit is involved in insulin binding. In this way,activation of the insulin receptor is believed to involvecross-connection of the alpha-subunits by insulin. A similar mechanismmay operate for IGF-1R, but one of the receptor binding interactionsappears to be different (Schäffer, 1994, Eur. J. Biochem.221:1127-1132).

The identification of molecular structures having a high degree ofspecificity for one or the other receptor is important to developingefficacious and safe therapeutics. For example, a molecule developed asan insulin agonist should have little or no IGF-1 activity in order toavoid the mitogenic activity of IGF-1 and a potential for facilitatingneoplastic growth. It is therefore important to determine whetherinsulin and IGF-1 share common three-dimensional structures but whichhave sufficient differences to confer selectivity for their respectivereceptors. Similarly, it would be desirable to identify other molecularstructures that mimic the active binding regions of insulin and/or IGF-1and which impart selective agonist or antagonist activity.

Although certain proteins are important drugs, their use as therapeuticspresents several difficult problems, including the high cost ofproduction and formulation, administration usually via injection andlimited stability in the bloodstream. Therefore, replacing proteins,including insulin or IGF-1, with small molecular weight drugs hasreceived much attention. However, to date, none of these efforts hasresulted in finding an effective drug replacement.

Peptides mimicking functions of protein hormones have been previouslyreported. Yanofsky et al. (1996, Proc. Natl. Acad. Sci. USA93:7381-7386) reported the isolation of a monomer antagonistic to IL-1with nanomolar affinity for the IL-1 receptor. This effort requiredconstruction and use of many phage displayed peptide libraries andsophisticated phage-panning procedures.

Wrighton et al. (1996, Science 273:458-464) and Livnah et al. (1996,Science 273:464-471) reported dimer peptides that bind to theerythropoietin (EPO) receptor with full agonistic activity in vivo.These peptides are cyclical and have intra-peptide disulfide bonds; likethe IL-1 receptor antagonist, they show no significant sequence identityto the natural ligand. Importantly, X-ray crystallography revealed thatit was the spontaneous formation of non-covalent peptide homodimerpeptides that enabled the dimerization two EPO receptors.

WO 96/04557 reported the identification of peptides and antibodies thatbound to active sites of biological targets, which were subsequentlyused in competition assays to identify small molecules that acted asagonist or antagonists at the biological targets. Renchler et al. (1994,Proc. Natl. Acad. Sci. USA 91:3623-3627) reported synthetic peptideligands of the antigen binding receptor that induced programmed celldeath in human B-cell lymphoma.

Most recently, Cwirla et al. (1997, Science 276:1696-1698) reported theidentification of two families of peptides that bound to the humanthrombopoietin (TPO) receptor and were competed by the binding of thenatural TPO ligand. The peptide with the highest affinity, whendimerized by chemical means proved to be as potent an in vivo agonist asTPO, the natural ligand.

V. SUMMARY OF THE INVENTION

This invention relates to the identification of amino acid sequencesthat specifically recognize sites involved in IR or IGF-1R activation.Specific amino acid sequences are identified and their agonist orantagonist activity at IR and/or IGF-1R has been determined. Suchsequences may be developed as potential therapeutics or as leadcompounds to develop other more efficacious ones. In addition, thesesequences may be used in high-throughput screens to identify and provideinformation on small molecules that bind at these sites and mimic orantagonize the functions of insulin or IGF-1. Furthermore, the peptidesequences provided by this invention can be used to design secondarypeptide libraries, which can be used to identify sequence variants thatincrease or otherwise modulate the binding and/or activity of theoriginal peptide at IR or IGF-1R. The peptide sequences of the inventioncan also be combined to make dimer or other multimeric peptides, whichcan be used for screening, diagnostic, and therapeutic applications asdescribed herein.

In one aspect of this invention, large numbers of peptides have beenscreened for their IR and IGF-1R binding and activity characteristics.Analysis of their amino acid sequences has identified certain consensussequences which may be used themselves or as core sequences in largeramino acid sequences conferring upon them agonist or antagonistactivity. Several generic amino acid sequences are disclosed which bindIR and/or IGF-1R with varying degrees of agonist or antagonist activitydepending on the specific sequence of the various peptides identifiedwithin each motif group. Also provided are amino or carboxyl terminalextensions capable of modifying the affinity and/or pharmacologicalactivity of the consensus sequences when part of a larger amino acidsequence. Further provided are peptides containing more than oneconsensus sequence (e.g., dimer peptides).

The amino acid sequences of this invention which bind IR and/or IGF-1Rinclude:

a. X₁ X₂ X₃ X₄ X₅ wherein X₁, X₂, X₄ and X₅ are aromatic amino acids,and X₃ is any polar amino acid (Formula 1; Group 1; A6 motif);

b. X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ wherein X₆ and X₇ are aromatic aminoacids, X₈, X₉, X₁₁ and X₁₂ are any amino acid, and X₁₀ and X₁₃ arehydrophobic amino acids (Formula 2; Group 3; B6 motif);

c. X₁₄ X₁₅ X₁₆ X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ wherein X₁₄, and X₁₇ are hydrophobicamino acids, X₁₅, X₁₆, X₁₈ and X₁₉ are any amino acid, and X₂₀ and X₂₁are aromatic amino acids (Formula 3; reverse B6; revB6).

d. X₂₂ X₂₃ X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ X₂₉ X₃₀ X₃₁ X₃₂ X₃₃ X₃₄ X₃₅ X₃₆ X₃₇ X₃₈X₃₉ X₄₀ X₄₁ wherein X₂₂, X₂₅, X₂₈, X₂₉, X₃₀, X₃₃, X₃₄, X₃₅, X₃₆, X₃₇,X₃₈, X₄₀, and X₄₁ are any amino acid, X₃₅ and X₃₇ may be any amino acidfor binding to IR, whereas X₃₅ is preferably a hydrophobic amino acidand X₃₇ is preferably glycine for binding to IGF-1R and possess agonistor antagonist activity. X₂₃ and X₂₆ are hydrophobic amino acids. Thissequence further comprises at least two cysteine residues, preferably atX₂₅ and X₄₀ X₃₁ and X₃₂ are small amino acids (Formula 4; Group 7; E8motif).

e. X₄₂ X₄₃ X₄₄ X₄₅ X₄₆ X₄₇ X₄₈ X₄₉ X₅₀ X₅₁ X₅₂ X₅₃ X₅₄ X₅₅ X₅₆ X₅₇ X₅₈X₅₉ X₆₀ X₆₁ wherein X₄₂, X₄₃, X₄₄, X₄₅, X₅₃, X₅₅, X₅₆, X₅₈, X₆₀ and X₆₁may be any amino acid, X₄₃, X₄₆, X₄₉, X₅₀, X₅₄ are hydrophobic aminoacids, X₄₇ and X₅₉ are preferably cysteines, X₄₈ is a polar amino acid,and X₅₁, X₅₂ and X₅₇ are small amino acids (Formula 5; mini F8 motif).

f. X₆₂ X₆₃ X₆₄ X₆₅ X₆₆ X₆₇ X₆₈ X₆₉ X₇₀ X₇₁ X₇₂ X₇₃ X₇₄ X₇₅ X₇₆ X₇₇ X₇₈X₇₉ _(X80) X₈₁ wherein X₆₂, X₆₅, X₆₈, X₆₉, X₇₁, X₇₃, X₇₆, X₇₇, X₇₈, X₈₀,and X₈₁ may be any amino acid; X₆₃, X₇₀, X₇₄ are hydrophobic aminoacids; X₆₄ is a polar amino acid, X₆₇ and X₇₅ are aromatic amino acidsand X₇₂ and X₇₉ are preferably cysteines capable of forming a loop(Formula 6; Group 2; D8 motif).

g. H X₈₂ X₈₃ X₈₄ X₈₅ X₈₆ X₈₇ X₈₈ X₈₉ X₉₀ X₉₁ X₉₂ wherein X₈₂ is prolineor alanine, X₈₃ is a small amino acid, X₈₄ is selected from leucine,serine or threonine, X₈₅ is a polar amino acid, X₈₆, X₈₈, X₈₉ and X₉₀are any amino acid, and X₈₇, X₉₁ and X₉₂ are an aliphatic amino acid(Formula 7).

h. X₁₀₄ X₁₀₅ X₁₀₆ X₁₀₇ X₁₀₈ X₁₀₉ X₁₁₀ X₁₁₁ X₁₁₂ X₁₁₃ X₁₁₄ wherein atleast one of the amino acids of X₁₀₆ through X₁₁₁, and preferably two,are tryptophan separated by three amino acids, and wherein at least oneof X₁₀₄, X₁₀₅ and X₁₀₆ and at least one of X₁₁₂, X₁₁₃ and X₁₁₄ arecysteine (Formula 8); and

i. an amino acid sequence comprising the sequence JBA5:DYKDLCQSWGVRIGWLAGLCPKK (SEQ ID NO:1541) or JBA5 minus FLAG® tag andterminal lysines: LCQSWGVRIGWLAGLCP (SEQ ID NO:1542) (Formula 9).

j. W X₁₂₃ G Y X₁₂₄ W X₁₂₅ X₁₂₆ (SEQ ID NO:1543) wherein X₁₂₃ is selectedfrom proline, glycine, serine, arginine, alanine or leucine, but morepreferably proline; X₁₂₄ is any amino acid, but preferably a charged oraromatic amino acid; X₁₂₅ is a hydrophobic amino acid preferably leucineor phenylalanine, and most preferably leucine. X₁₂₆ is any amino acid,but preferably a small amino acid (Formula 10; Group 6 motif).

In one embodiment, peptides comprising a preferred amino acid sequenceFYX₃ WF (SEQ ID NO:1544) (Formula 1; Group 1; A6 motif) have beenidentified which competitively bind to sites on IR. Surprisingly,peptides comprising an amino acid sequence FYX₃ WF (SEQ ID NO:1544) canpossess agonist or antagonist activity at IR or IGF-1R.

This invention also identifies at least two distinct binding sites on IRand IGF-1R (Site 1 and Site 2) based on the differing ability of certainof the peptides to compete with one another and ligand for binding to IRor IGF-1R. Accordingly, this invention provides amino acid sequencesthat bind specifically to one or both sites of IR or IGF-1R.Furthermore, specific amino acid sequences are provided which haveagonist or antagonist characteristics based on their ability to bind tothe specific sites of IR or IGF-1R.

In another embodiment of this invention, amino acid sequences which bindto one or more sites of IR or IGF-1R (e.g., Site 1 or Site 2) arecovalently linked together to form multivalent ligands. Thesemultivalent ligands are capable of forming complexes with a plurality ofIR or IGF-1R. Either the same or different amino acid sequences arecovalently bound together to form homo- or heterocomplexes.

In various aspects of the invention, monomer subunits are covalentlylinked at their N-termini or C-termini to form N-N, C-C, N-C, or C-Nlinked dimer peptides. In one example, dimer peptides are used to formreceptor complexes bound through the same corresponding sites, e.g.,Site 1-Site 1 or Site 2-Site 2 dimers. Alternatively, heterodimerpeptides are used to bind to different sites on one receptor or to causereceptor complexing through different sites, e.g., Site 1-Site 2 or Site2-Site 1 dimers. In one novel aspect of the invention, Site 2-Site 1dimers find use as insulin agonists, while certain Site 1-Site 2 dimersfind use as insulin antagonists.

In various embodiments, insulin agonists comprise Site 1-Site 1 dimerpeptide sequences S325, S332, S333, S335, S337, S353, S374-S376, S378,S379, S381, S414, S415, and S418; whereas other insulin agonistscomprise Site 2-Site 1 dimer peptide sequences S455, S457, S458, S467,S468, S471, S499, S510, S518, S519, and S520, as described herein below.In one preferred embodiment, an insulin agonist comprises the sequenceof the S519 dimer peptide, which shows insulin-like activity in both invitro and in vivo assays.

The present invention also provides assays for identifying compoundsthat mimic the binding characteristics of insulin or IGF-1. Suchcompounds may act as antagonists or agonists of insulin or IGF-1function in cell based assays.

This invention further provides kits for identifying compounds that bindto IR and/or IGF-1R. Also provided are therapeutic compounds that bindthe insulin receptor or the IGF-1 receptor.

Other embodiments of this invention are the nucleic acid sequencesencoding the amino acid sequences of the invention. Also within thescope of this invention are vectors containing the nucleic acids andhost cells which express the nucleic acids encoding the amino acidsequences which bind at IR and/or IGF-1R and possess agonist orantagonist activity.

This invention also provides amino acid sequences that bind to activesites of IR and/or IGF-1R and to identify structural criteria forconferring agonist or antagonist activity at IR or IGF-1R.

This invention further provides specific amino acid sequences thatpossess agonist, partial agonist, or antagonist activity at either IR orIGF-1R. Such amino acid sequences are potentially useful as therapeuticsthemselves or may be used to identify other molecules, especially smallorganic molecules, which possess agonist or antagonist activity at IR orIGF-1R.

In addition, the present invention provides structural informationderived from the amino acid sequences of this invention, which may beused to construct other molecules possessing the desired activity at therelevant IR binding site.

VI. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1O; 2A-2E; 3A-3E; 4A-4I; 43A-43B, 44A-44B: Amino acid sequencesidentified by panning peptide libraries against IGF-1R and/or IR. Theamino acids are represented by their one-letter abbreviation. The ratiosover background are determined by dividing the signal at 405 nm (E-Tag,IGF-1R, or IR) by the signal at 405 nm for non-fat milk. The IGF-1R/IRRatio Comparison is determined by dividing the ratio of IGF-1R by theratio of IR. The IR/IGF-1R Ratio Comparison is determined by dividingthe ratio of IR by the ratio of IGF-1R. HIT indicates binder; CANDindicates binder candidate; LDH indicates binding to lactatedehydrogenase (negative control); Sp/Irr indicates the ratio of specificbinding over non-specific binding.

The design of each library is shown in the first line in bold. In thedesign, symbol ‘X’ indicates a random position, an underlined amino acidindicates a doped position at the nucleotide level, and other positionsare held constant. Additional abbreviations in the B6H library are: ‘O’indicates an NGY codon where Y is C or T; ‘J’ indicates an RHR codonwhere R is A or G, and H is A, C, or T; and ‘U’ indicates an VVY codonwhere V is A, C, or G, and Y is C or T. The ‘h’ in the 20E2 librariesindicates an NTN codon.

Symbols in the listed sequences include: Q indicates a positioncorresponding to a TAG stop codon; # indicates a position correspondingto a TAA stop codon; * indicates a position corresponding to a TGA stopcodon; and ? indicates an unknown amino acid. It is believed that a Wreplaces the TGA stop codon when expressed. The Q residues representtranslation read-through at TAG stop codons. Except for the 20C and A6Llibraries, all libraries are designed with the short FLAG® epitope DYKD(SEQ ID NO:1545; Hopp et al., 1988, Bio/Technology 6:1205-1210) at theN-terminus of the listed sequence and AAAGAP (SEQ ID NO:1546) at theC-terminus. The 20C and A6L libraries have the full length FLAG® epitopeDYKDDDDK (SEQ ID NO:1547).

FIG. 1A: Formula 1 motif peptide sequences obtained from a random 40merlibrary panned against IR (SEQ ID NOS:1-3).

FIG. 1B: Formula 1 motif peptide sequence obtained from a random 40merlibrary panned against IGF-1R (SEQ ID NOS:4-6).

FIG. 1C: Formula 1 motif peptide sequences obtained from a random 20merlibrary panned against IR (SEQ ID NOS:7-29).

FIG. 1D: Formula 1 motif peptide sequences obtained from a random 20merlibrary panned against IGF-1R (SEQ ID NOS:30-33).

FIG. 1E: Formula 1 motif peptide sequences obtained from a 21mer libraryconstructed to contain X₁₋₁₀NFYDWFVX₁₈₋₂₁ (SEQ ID NO:34; also referredto as “A6S”) panned against IR (SEQ ID NOS:35-98).

FIG. 1F: Formula 1 motif peptide sequences obtained from a 21mer libraryconstructed to contain X₁₋₁₀NFYDWFVX₁₈₋₂₁ (SEQ ID NO:34; also referredto as “A6S”) panned against IGF-1R (SEQ ID NOS:99-166).

FIG. 1G: Formula 1 motif peptide sequences obtained from a libraryconstructed to contain variations outside the consensus core of the A6peptide as indicated (referred to as “A6L” (SEQ ID NO:167)) pannedagainst IR (SEQ ID NOS:168-216).

FIG. 1H: Formula 1 motif peptide sequences obtained from a libraryconstructed to contain variations outside the consensus core of the A6peptide as indicated (referred to as “A6L” (SEQ ID NO:167)) pannedagainst IGF-1R (SEQ ID NOS:217-244).

FIG. 1I: Formula 1 motif peptide sequences obtained from a libraryconstructed to contain variations in the consensus core of the E4Dpeptide (SEQ ID NO:245) (as indicated) panned against IR (SEQ IDNOS:246-305).

FIG. 1J: Formula 1 motif peptide sequences obtained from a libraryconstructed to contain variations in the consensus core of the E4Dpeptide (SEQ ID NO:245) (as indicated) panned against IGF-1R (SEQ IDNOS:306-342).

FIG. 1K: Formula 1 motif peptide sequences obtained from a libraryconstructed using the sequence X₁₋₆FHENFYDWFVRQVSX₂₁₋₂₆ (SEQ ID NO:343;H2C-A) panned against IR (SEQ ID NOS:344-430).

FIG. 1L: Formula 1 motif peptide sequences obtained from a libraryconstructed using the sequence X₁₋₆FHENFYDWFVRQVSX₂₁₋₂₆ (SEQ ID NO:343;H2C-A) panned against IGF-1R (SEQ ID NOS:431-467).

FIG. 1M: Formula 1 motif peptide sequences obtained from a libraryconstructed using the sequence X₁₋₆FHXXFYXWFX₁₆₋₂₁ (SEQ ID NO:468;H2C-B) and panned against IR (SEQ ID NOS:469-575).

FIG. 1N: Formula 1 motif peptide sequences obtained from a libraryconstructed using the sequence X₁₋₆FHXXFYXWFX₁₆₋₂₁ (SEQ ID NO:468;H2C-B) and panned against IGF-1R (SEQ ID NOS:576-657).

FIG. 1O: Formula 1 motif peptide sequences obtained from other librariespanned against IR (SEQ ID NOS:658-712).

FIG. 2A: Formula 4 motif peptide sequences identified from a random20mer library panned against IR (SEQ ID NO:713).

FIG. 2B: Formula 4 motif peptide sequences identified from a libraryconstructed to contain variations in the F8 peptide (SEQ ID NO:713) asindicated (15% dope; referred to as “F815”) panned against IR (SEQ IDNOS:714-796).

FIG. 2C: Formula 4 motif peptide sequences identified from a libraryconstructed to contain variations in the F8 peptide (SEQ ID NO:713) asindicated (15% dope; referred to as “F815”) panned against IGF-1R (SEQID NOS:797-811).

FIG. 2D: Formula 4 motif peptide sequences identified from a libraryconstructed to contain variations in the F8 peptide (SEQ ID NO:713) asindicated (20% dope; referred to as “F820”) panned against IR (SEQ IDNOS:812-861).

FIG. 2E: Formula 4 motif peptide sequences identified from otherlibraries panned against IR (SEQ ID NOS:862-925).

FIG. 3A: Formula 6 motif peptide sequences identified from a random20mer library and panned against IR (SEQ ID NOS:926-928).

FIG. 3B: Formula 6 motif peptide sequences identified from a libraryconstructed to contain variations in the D8 peptide (SEQ ID NO:929) asindicated (15% dope; referred to as “D815”) panned against IR (SEQ IDNOS:930-967).

FIG. 3C: Formula 6 motif peptide sequences identified from a libraryconstructed to contain variations in the D8 peptide (SEQ ID NO:929) asindicated (20% dope; referred to as “D820”) panned against IR (SEQ IDNOS:968-1010).

FIG. 3D: Formula 6 motif peptide sequences identified from a libraryconstructed to contain variations in the D8 peptide (SEQ ID NO:929) asindicated (20% dope; referred to as “D820”) panned against IGF-1R (SEQID NOS:1011-1059).

FIG. 3E: Formula 6 motif peptide sequences identified from otherlibraries panned against IR (SEQ ID NOS:1060-1061).

FIG. 4A: Formula 10 motif peptide sequences identified from random 20merlibraries panned against IGF-1R (SEQ ID NOS:1062-1077).

FIG. 4B: Formula 10 motif peptide sequences identified from random 20merlibraries panned against IR (SEQ ID NOS:1078-1082).

FIG. 4C: Miscellaneous peptide sequences identified from a random 20merlibrary panned against IR (SEQ ID NOS:1083-1086).

FIG. 4D: Miscellaneous peptide sequences identified from a random 40merlibrary panned against IR (SEQ ID NOS:1087-1088).

FIG. 4E: Miscellaneous peptide sequences identified from a random 20merlibrary panned against IGF-1R (SEQ ID NOS:1089-1092).

FIG. 4F: Miscellaneous peptide sequences identified from an X₁₋₄ C X₆₋₂₀library and panned against IGF-1R (SEQ ID NOS:1093-1113).

FIG. 4G: Miscellaneous peptide sequences identified from a libraryconstructed to contain variations of the F8 peptide (SEQ ID NO:1114) asindicated (F815) panned against IGF-1R (SEQ ID NOS:1115-1118).

FIG. 4H: Miscellaneous peptide sequences identified from a libraryconstructed to contain variations in the F8A11 peptide (SEQ ID NO:1119)as indicated (referred to as “NNKH”) panned against IR (SEQ IDNOS:1120-1142).

FIG. 4I: Miscellaneous peptide sequences identified from a libraryconstructed to contain variations in the F8A11 peptide (SEQ ID NO:1119)as indicated (referred to as “NNKH”) panned against IGF-1R (SEQ IDNOS:1143-1154).

FIG. 5A: Summary of specific representative amino acid sequences fromFormulas 1, 4, 6, and 10 (SEQ ID NOS:1155-1180).

FIG. 5B: Summary of specific representative amino acid sequences fromFormulas 1, 4, 6, and 10 (SEQ ID NOS:1181-1220).

FIG. 6: Illustration of 2 binding site domains on IR based oncompetition data.

FIG. 7: Schematic illustration of potential binding schemes to themultiple binding sites on IR.

FIG. 8: Biopanning results and sequence alignments of Group 1 ofIR-binding peptides (SEQ ID NOS:1221-1243). The number of sequencesfound is indicated on the right side of the figure together with data onthe phage binding to either IR or IGF-1R receptor. Absorbance signalsare indicated by: ++++, >30X over background; +++, 15-30X; ++, 5-15X; +,2-5X; and 0, <2X.

FIGS. 9A-9B: Biopanning results and sequence alignments of Groups 2, 6,and 7 of IR-binding peptides (SEQ ID NOS:1244-1261). The number ofsequences found is indicated on the right side of the figure togetherwith data on the phage binding to either IR or IGF-1R receptor.Absorbance signals are indicated by: ++++, >30X over background; +++,15-30X; ++, 5-15X; +, 2-5X; and 0, <2X.

FIGS. 10A-10C: Insulin competition data determined for various monomerand dimer peptides. FIG. 10A shows the competition curve. FIG. 10B showsthe symbol key for the peptides. FIG. 10C shows the description of thepeptides.

FIGS. 11A-11D: Insulin competition data determined for various monomerand dimer peptides. FIG. 11A shows the competition curve. FIG. 11B showsthe symbol key for the peptides. FIG. 11C shows the description of thepeptides. FIG. 11D shows IR binding affinity for the peptides.

FIGS. 12A-12D: Results of free fat cell assays for truncated syntheticRP9 monomer peptides, S390 and S394. FIG. 12A shows the results forpeptide S390. FIG. 12B shows the results for peptide S394. FIG. 12Cshows the amino acid sequence of peptides S390 and S394 (SEQ ID NOS:1794and 1788, respectively in order of appearance). FIG. 12D shows theresults for full-length RP9 peptide.

FIGS. 13A-13C: Results of free fat cell assays for truncated syntheticRP9 dimer peptides, S415 and S417. FIG. 13A shows the results forpeptide S415. FIG. 13B shows the results for peptide S417. FIG. 13Cshows the amino acid sequence of peptides S415 and S417 (SEQ IDNOS:1795-1796).

FIGS. 14A-14C: Results of free fat cell assays for RP9 homodimerpeptides, 521 and 535. FIG. 14A shows the results for peptide 521. FIG.14B shows the results for peptide 535. FIG. 14C shows the amino acidsequence of peptides 521 and 535.

FIGS. 15A-15C: Results of free fat cell assays for RP9-D8 heterodimerpeptides, 537 and 538. FIG. 15A shows the results for peptide 537. FIG.15B shows the results for peptide 538. FIG. 15C shows the amino acidsequence of peptides 537 and 538.

FIGS. 16A-16C: Results of free fat cell assays for RP9-D8 heterodimerpeptides 537 and 538. FIG. 16A shows the results for peptide 537. FIG.16B shows the results for peptide 538. FIG. 16C shows the amino acidsequence of peptides 537 and 538.

FIGS. 17A-17B: Results of free fat cell assays for D8-RP9 heterodimerpeptide, 539. FIG. 17A shows the results for peptide 539. FIG. 17B showsthe amino acid sequence of peptide 539.

FIGS. 18A-18D: Results of free fat cell assays for Site 1/Site 2 dimerpeptides with constituent monomer peptides with Site 1-Site 2 C-N (FIG.18A), Site 1-Site 2, N—N (FIG. 18B), Site 1-Site 2, C-C (FIG. 18C), andSite 2-Site 1, C-N (FIG. 18D) orientations and linkages, respectively.

FIGS. 19A-19B: Results of human insulin receptor kinase assays forvarious monomer and dimer peptides. FIG. 19A shows the substratephosphorylation curve.

FIG. 19B shows the EC₅₀ values.

FIGS. 20A-20B: Results of human insulin receptor kinase assays for Site1-Site 2 and Site 2-Site 1 dimer peptides. FIG. 20A shows the substratephosphorylation curve. FIG. 20B shows the EC₅₀ values.

FIGS. 21A-21B: Results of human insulin receptor kinase assays for Site1-Site 2 and Site 2-Site 1 peptides. FIG. 21A shows the substratephosphorylation curve. FIG. 21B shows the EC₅₀ values.

FIGS. 22A-22B: Results of time-resolved fluorescence resonance transferassays for assessing the ability of various monomer and dimer peptidesto compete with biotinylated RP9 monomer peptide for binding to solublehuman insulin receptor-immunoglobulin heavy chain chimera. FIG. 22Ashows the binding curve. FIG. 22B shows the symbol key and descriptionof the peptide sequences (SEQ ID NOS:2117, 1916-1917, 1558, 1994,1960-1961, 2008, 1794, 2015-2016, 1560, and 2001-2002, respectively inorder of appearance).

FIGS. 23A-23C: Results of time-resolved fluorescence resonance transferassays indicating the ability of various monomer and dimer peptide tocompete with biotinylated S175 monomer peptide or biotinylated RP9monomer peptide for binding to soluble human insulinreceptor-immunoglobulin heavy chain chimera. FIGS. 23A-23B show thebinding curves. FIG. 23C shows the symbol key and description of thepeptide sequences (SEQ ID NOS:2117, 1916-1917, 1558, 1994, 1960-1961,2008, 1794, 2015-2016, 1560, and 2001-2002, respectively in order ofappearance).

FIGS. 24A-24B: Results of fluorescence polarization assays indicatingthe ability of various monomer and dimer peptide to compete withfluoroscein labeled RP9 monomer peptide for binding to soluble humaninsulin receptor ectodomain. FIG. 24A shows the binding curve. FIG. 24Bshows the symbol key and description of the peptide sequences (SEQ IDNOS:2117, 1916-1917, 1558, 1994, 1960-1961, 2008, 1794, 2015-2016, 1560and 2001-2002, respectively in order of appearance).

FIGS. 25A-25B: Results of fluorescence polarization assays indicatingthe ability of various monomer and dimer peptides to compete withfluoroscein labeled RP9 monomer peptide for binding to soluble humaninsulin mini-receptor. FIG. 25A shows the binding curve. FIG. 25B showsthe symbol key and description of the peptide sequences (SEQ IDNOS:2117, 1916-1917, 1558, 1994, 1960-1961, 2008, 1794, 2015-2016, 1560,and 2001-2002, respectively in order of appearance).

FIGS. 26A-26B: Results of fluorescence polarization assays indicatingthe ability of various monomer and dimer peptides to compete withfluorescein labeled insulin for binding to soluble human insulinreceptor ectodomain. FIG. 26A shows the binding curve. FIG. 26B showsthe symbol key and description of the peptide sequences (SEQ IDNOS:2117, 1916-1917, 1558, 1994, 1960-1961, 2008, 1794, 2015-2016, 1560,and 2001-2002, respectively in order of appearance).

FIGS. 27A-27B: Results of fluorescence polarization assays indicatingthe ability of various monomer and dimer peptides to compete withfluorescein labeled insulin for binding to soluble human insulinmini-receptor. FIG. 27A shows the binding curve. FIG. 27B shows thesymbol key and description of the peptide sequences (SEQ ID NOS:2117,1916-1917, 1558, 1994, 1960-1961, 2008, 1794, 2015-2016, 1560, and2001-2002, respectively in order of appearance).

FIG. 28: A schematic drawing for the construction of protein fusions ofthe maltose binding protein.

FIG. 29: BIAcore analysis of competition binding between IR and maltosebinding protein fusion peptides H2C-9aa-H2C, H2C, and H2C-3aa-H2C.

FIG. 30: Stimulation of IR autophosphorylation in vivo by maltosebinding protein fusion peptides.

FIGS. 31A-31C: Results of free fat cell assays for insulin and Site2-Site 1 peptides, S519 and S520. FIG. 31A shows the results for S519.FIG. 31B shows the results for S520. FIG. 31C shows the EC₅₀ values.

FIGS. 32A-32B: Results of human insulin receptor kinase assays forinsulin and Site 2-Site 1 peptides S519 and S520. FIG. 32A shows thesubstrate phosphorylation curve. FIG. 32B shows the calculated Bestfitvalues.

FIG. 33: Results of in vivo experiments showing the effect ofintravenous administration of Site 2-Site 1 peptide S519 in Wistar rats:

FIGS. 34A-34E: Results of phage competition studies with IGF-1 peptidesRP9 (Site 1) and D815 (Site 2). Phage: RP9 (A6-like); RP6 (B6-like);D8B12 (Site 2); and D815 (Site 2); Peptides: RP9 and D815. FIGS. 34A-34Bshow the competition curves. FIGS. 34C-34E show the symbol keys andpeptide groups.

FIG. 35A-35E: Phage competition studies with Site 2-Site 1 dimerpeptides containing 6- or 12-amino acid linkers. Phage: RP9, RP6, D8B12,and D815; Peptides: D815-6L-RP9 and D815-12L-RP9. FIGS. 35A-35B show thecompetition curves. FIGS. 35C-35E show the symbol keys and peptidegroups.

FIG. 36: Results of IGF-1 agonist assay using FDC-P2 cells. Site 1peptides RP6, RP9, G33, and Site 2 peptide D815 were tested in theagonist assay.

FIG. 37: Results of IGF-1 antagonist assay using FDCP-2 cells. Site 1peptides RP6, RP9, G33, and Site 2 peptide D815 were tested in theantagonist assay.

FIG. 38: Results of IGF-1 agonist assay using FDCP-2 cells. Site 1peptides 20E2, S175, and RP9 were tested in the agonist assay.

FIG. 39: Results of agonist and antagonist studies with peptide monomersand dimers. Monomers: D815 and RP9; Dimers: D815-6aa-RP9 andD815-12aa-RP9.

FIG. 40: Results of agonist and antagonist studies with peptide monomersand dimers. Monomers: G33 and D815; Dimer: D815-6aa-G33.

FIG. 41: Results of agonist and antagonist studies with peptide monomersand dimers. Monomers: G33, D815 and RP9; Dimers: D815-6aa-RP9 andD815-12aa-RP9.

FIG. 42: IGF-1 standard curve using FDC-P2 cells.

FIGS. 43A-43B: Peptide monomers identified from G33 and RP6 secondarylibraries panned against IGF-1R (SEQ ID NOS:1262-1432). FIG. 43A showspeptides from G33 secondary library; FIG. 43B shows peptides from RP6secondary library.

FIGS. 44A-44B: Peptide dimers identified from libraries panned againstIR or IGF-1R (SEQ ID NOS:1433-1540). FIG. 44A shows dimer peptidespanned against IR; FIG. 44B shows dimer peptides panned against IGF-1R.

FIG. 45: Results of heterogeneous time-resolved fluorometric assaysshowing the effect of recombinant peptide G33 (rG33) on the binding ofbiotinylated-recombinant human IGF-1 (b-rhIGF-1) to recombinant humanIGF-1R (rhIGF-1R).

FIG. 46: Results of heterogeneous time-resolved fluorometric assaysshowing the effect of recombinant peptide D815 (rD815) on the binding ofbiotinylated-recombinant human IGF-1 (b-rhIGF-1) to recombinant humanIGF-1R (rhIGF-1R).

FIG. 47: Results of heterogeneous time-resolved fluorometric assaysshowing the effect of recombinant peptide RP9 on the binding ofbiotinylated-recombinant human IGF-1 (b-rhIGF-1) to recombinant humanIGF-1R (rhIGF-1R).

FIG. 48: Results of heterogeneous time-resolved fluorometric assayshowing the effect of recombinant peptide D815-6-G33 on the binding ofbiotinylated-recombinant human IGF-1 (b-rhIGF-1) to recombinant humanIGF-1R (rhIGF-1R).

FIG. 49: Results of heterogeneous time-resolved fluorometric assaysshowing the effect of recombinant peptide D815-6-RP9 on the binding ofbiotinylated-recombinant human IGF-1 (b-rhIGF-1) to recombinant humanIGF-1R (rhIGF-1R).

FIG. 50: Results of heterogeneous time-resolved fluorometric assaysshowing the effect of recombinant peptide D815-12-RP9 on the binding ofbiotinylated-recombinant human IGF-1 (b-rhIGF-1) to recombinant humanIGF-1R (rhIGF-1R).

FIG. 51: Results of heterogeneous time-resolved fluorometric assaysshowing the effect of IGF-1 on the binding of biotinylated-recombinanthuman IGF-1 (b-rhIGF-1) to recombinant human IGF-1R (rhIGF-1R).

FIG. 52: Results of time-resolved fluorescence resonance energy transferassays showing the effect of Site 1 peptides, Site 2 peptides, andrhIGF-1 on the dissociation of biotinylated-20E2 (b-20E2, Site 1) fromrecombinant human IGF-1R.

FIG. 53: Results of time-resolved fluorescence resonance energy transferassays showing the effect of various peptide monomers and dimers on thedissociation of biotinylated-20E2 (b-20E2, Site 1) from recombinanthuman IGF-1R.

FIGS. 54A-54B, 55A-55B, 56A-56B, 57A-57B, 58A-58B, 59A-59B, 60A-60C,61A-61B, 62A-62B, 63A-63B, and 64A-64B: Amino acid sequences identifiedby panning peptide libraries against IGF-1R. The amino acids arerepresented by their one-letter abbreviation. The ratios over backgroundare determined by dividing the signal at 405 nm (E-Tag, IGF-1R, or IR)by the signal at 405 nm for non-fat milk. The IGF-1R/IR ratio comparisonis determined by dividing the ratio of IGF-1R by the ratio of IR. TheIR/IGF-1R ratio comparison is determined by dividing the ratio of IR bythe ratio of IGF-1R. Sp/Irr=the ratio of specific binding overnon-specific binding; LDH=lactate dehydrogenase (negative control).

Where included, the design of each library is shown in the first line inbold. In the design, symbol ‘X’ indicates a random position, anunderlined amino acid indicates a doped position at the nucleotidelevel, and other positions are held constant. Symbols in the listedsequences include: Q indicates a position corresponding to a TAG stopcodon; # indicates a position corresponding to a TAA stop codon; *indicates a position corresponding to a TGA stop codon; and ? indicatesan unknown amino acid. The Q residues represent translation read-throughat TAG stop codons. All libraries were designed with the short FLAG®Epitope DYKD (SEQ ID NO:1545; Hopp et al., 1988, Bio/Technology6:1205-1210) at the N-terminus of the listed sequence and an E-tagepitope (GAPVPYPDPLEPR; SEQ ID NO:XX) at the C-terminus.

FIGS. 54A-54B: Peptides identified from a RP6 secondary library pannedagainst IGF-1R. The RP9 peptide is a Formula 1, Site 1 monomer.

FIGS. 55A-55B: Peptides identified from a RP9-NPB25 secondary librarypanned against IGF-1R. The RP9-NPB25 peptide is a Formula 2, Site 1monomer with a 25 amino acid C-terminal extension.

FIGS. 56A-56B: Peptides identified from a RP30-IGF-NPB20 secondarylibrary panned against IGF-1R. The RP30-IGF-NPB20 peptide is a Site 1,Formula 2 monomer with a 20 amino acid C-terminal extension.

FIGS. 57A-57B: Peptides identified from a NPB20-RP30-IGF secondarylibrary panned against IGF-1R. The NPB20-RP30-IGF peptide is a Site 1,Formula 2 monomer with a 20 amino acid N-terminal extension.

FIGS. 58A-58B: Peptides identified from a D815 secondary library pannedagainst IGF-1R. The D815 peptide is a Formula 6, Site 2 monomer.

FIGS. 59A-59B: Peptides identified from a RP6-D815 secondary librarypanned against IGF-1R. The RP6-D815 peptide is a Site 1-Site 2 dimerwith no linker.

FIGS. 60A-60C: Peptides identified from a RP6-6aa-D815 secondary librarypanned against IGF-1R. The RP6-6aa-D815 peptide is a Site 1-Site 2 dimerwith a 6 amino acid linker.

FIGS. 61A-61B: Peptides identified from a RP6-RP9 secondary librarypanned against IGF-1R. The RP6-RP9 peptide is a Site 1-Site 1 dimer withno linker.

FIGS. 62A-62B: Peptides identified from a RP6-6aa-RP9 secondary librarypanned against IGF-1R. The RP6-6aa-RP9 peptide is a Site 1-Site 1 dimerwith a 6 amino acid linker.

FIGS. 63A-63B: Peptides identified from a D815-RP6 secondary librarypanned against IGF-1R. The D815-RP6 peptide is a Site 2-Site 1 dimerwith no linker.

FIGS. 64A-64B: Peptides identified from a D815-6aa-RP6 secondary librarypanned against IGF-1R. The D815-6aa-RP6 peptide is a Site 2-Site 1 dimerwith a 6 amino acid linker.

FIGS. 65A-65F: Dose related increase in cell proliferation of MiaPaCaand MCF-7 cells as measured in response to IGF-1, IGF-2, and insulin.Cells were treated with either IGF-1, IGF-2, or insulin. FIG. 65A:Results for MiaPaCa cells incubated with IGF-1; FIG. 65B: MiaPaCa cellsincubated with IGF-2; FIG. 65C: MiaPaCa cells incubated with insulin;FIG. 65D: MCF-7 cells incubated with IGF-1; FIG. 65E: MCF-7 cellsincubated with IGF-2; FIG. 65F: MCF-7 cells incubated with insulin.

FIGS. 66A-66C: Peptide RP33-IGF competes with IGF-1 binding for bindingto IGF-1R and antagonizes receptor activity in cell-based assays. Forcompetition experiments, the ALPHAScreen assay format was used (seebelow). For antagonism assays, RP33-IGF was added to cells, cells wereincubated with IGF-1, and cell number was determined. FIG. 66A:Inhibition of IGF-1 binding as a function of RP33-IGF concentration.FIG. 66B: Antagonism of IGF-1R in MCF-7 cells by peptide RP33-IGF. FIG.66C: Antagonism of IGF-1R in MiaPaCa cells by peptide RP33-IGF.

FIGS. 67A-67B: IGF-1 stimulates transient phosphorylation of IRS-1 inMCF-7 cells. Cells were stimulated with IGF-1 for 0, 2, 10, 30, 60minutes and total protein was immunoprecipitated for each analysis. FIG.67A: Western blot analysis of endogenous IRS-1; FIG. 67B: Western blotanalysis of phosphorylated IRS-1; Lane 1: No addition; Lane 2: 2 minutetime point; Lane 3: 10 minute time point; Lane 4: 30 minute time point;Lane 5: 60 minute time point.

FIGS. 68A-68B: Phosphorylation of IRS-1 in MCF-7 cells induced by IGF-1is dose-dependant. Cells were exposed to increasing concentrations ofIGF-1 and total protein was immunoprecipitated. Stimulation by 0.50 nMIGF-1 resulted in a sub-maximal level of phosphorylation that wasconsistently visualized in Western blot analysis. FIG. 68A: Western blotanalysis of endogenous IRS-1; FIG. 68B: Western blot analysis ofphosphorylated IRS-1; Lane 1: No addition; Lane 2: 0.05 nM IGF-1; Lane3: 0.1 nM IGF-1; Lane 4: 5 nM IGF-1; Lane 5: 1 nM IGF-1; Lane 6: 0.5 nMIGF-1; Lane 7: 10 nM IGF-1; Lane 8: 50 nM IGF-1.

FIGS. 69A-69B: Blockade of IGF-1-induced phosphorylation of IRS-1 inMCF-7 cells by synthetic peptides RP6KK and RP33-IGF. Unrelated peptidesKCB1 (VSIGECGGLRHHRVRELCLV; SEQ ID NO:XX) and DGI3-D1(ECRWFRPWRCPGLLSTGGGR; SEQ ID NO:XX) were used as negative controls.FIG. 69A: Western blot analysis of expressed IRS-1; FIG. 69B: Westernblot analysis of phosphorylated IRS-1. Lane 1: no addition; Lane 2:DGI3-D1; Lane 3: KCB1; Lane 4: IGF-1 plus DGI3-D1; Lane 5: IGF-1 plusKCB1; Lane 6: IGF-1 plus RP6KK; Lane 7: IGF-1 plus RP33-IGF; Lane 8:IGF-1.

FIGS. 70A-70C: Peptides RP54 and RP52 compete with IGF-1 for binding toIGF-1R, and act as antagonists in cell proliferation assays. Forantagonism assays, RP54 or RP52 was added to cells, cells were incubatedwith IGF-1, and cell number was determined. FIG. 70A: Antagonism ofIGF-1R by RP54 in MCF-7 cells; FIG. 70B: Antagonism of RP54 in MiaPaCacells. FIG. 70C: Antagonism of IGF-1 by RP52 in MCF-7 cells.

FIGS. 71A-71F: Peptide monomers with IGF-1R agonist or antagonistactivity in MCF-7 or MiaPaCa cell proliferation assays compete againstIGF-1 for binding to IGF-1R. Potencies of peptide competition weredetermined using the AlphaScreen assay format (see below). FIG. 71A:RP60 peptide; FIG. 71B: RP48 peptide; FIG. 71C: sG33 peptide; FIG. 71D:C1 peptide; FIG. 71E: L-RP9ex peptide; FIG. 71F: 12-RP9ex peptide.

FIGS. 72A-72E: Peptide dimers with IGF-1R agonist activity in MCF-7 orMiaPaCa cell proliferation assays compete with IGF-1 for binding toIGF-1R. Potencies of peptide competition were determined using theAlphaScreen assay format (see below). FIG. 72A: rRP30-IGF-12-D112peptide (Site 1-Site 1); FIG. 72B: rRP30-IGF-12-RP31-IGF peptide (Site1-Site 2); FIG. 72C: rRP31-IGF-12-RP30-IGF peptide (Site 2-Site 1); FIG.72D: rD112-12-RP30-IGF peptide (Site 1-Site 1); FIG. 72E: rD112-12-D112peptide (Site 1-Site 1).

FIGS. 73A-73D: Peptide monomers with IGF-1R agonist activity in MCF-7 orMiaPaCa cell proliferation assays. FIG. 73A: RP60 peptide; FIG. 73B:RP48 peptide; FIG. 73C: G33 peptide; FIG. 73D: L-RP9ex peptide.

FIGS. 74A-74I: Peptide dimers with IGF-1R agonist activity in MCF-7 orMiaPaCa cell proliferation assays. FIG. 74A: RP30-IGF-12-D112 (Site1-Site 1); FIG. 74B: RP30-IGF-12-RP31-IGF (Site 1-Site 2); FIG. 74C:RP31-IGF-12-RP30-IGF (Site 2-Site 1); FIG. 74D: D112-12-RP30-IGF (Site1-Site 1); FIG. 74E: RP6-L-D8B12 (Site 1-Site 2); FIG. 74F: D8B12-12-RP9(Site 2-Site 1); FIG. 74G: D112-12-D112 (Site 1-Site 1); FIG. 74H:RP9-12-RP9 (Site 1-Site 1); FIG. 74I: RP9-L-RP6 (Site 1-Site 1).

VII. DETAILED DESCRIPTION OF THE INVENTION

This invention relates to amino acid sequences comprising motifs thatbind to the insulin receptor (IR) and/or insulin-like growth factorreceptor (IGF-1R). In addition to binding to IR and/or IGF-1R, the aminoacid sequences also possess either agonist, partial agonist orantagonist activity at IR or IGF-1R. In addition, the amino acidsequences bind to separate binding sites (Sites 1 or 2) on IR or IGF-1R.

Although capable of binding to IR or IGF-1R at sites which participatein conferring agonist or antagonist activity, the amino acid sequencesare not based on the native insulin or IGF-1 sequences, nor do theyreflect an obvious homology to any such sequences.

The amino acid sequences of the invention may be peptides, polypeptides,or proteins. These terms as used herein should not be consideredlimiting with respect to the size of the various amino acid sequencesreferred to herein and which are encompassed within this invention.Thus, any amino acid sequence comprising at least one of the IR orIGF-1R binding motifs disclosed herein, and which binds to IR or IGF-1Ris within the scope of this invention. In preferred embodiments, theamino acid sequences confer insulin or IGF-1 agonist or antagonistactivity. The amino acid sequences of the invention are typicallyartificial, i.e., non-naturally occurring peptides, polypeptides, orfragments thereof. The amino acid sequences of the invention do notinclude insulin, insulin-like growth factors, antibodies against insulinreceptors or insulin-like growth factor receptors, or fragments thereof.Amino acid sequences useful in the invention may be obtained throughvarious means such as chemical synthesis, phage display, cleavage ofproteins or polypeptides into fragments, or by any means which aminoacid sequences of sufficient length to possess binding ability may bemade or obtained.

The amino acid sequences provided by this invention should have anaffinity for IR sufficient to provide adequate binding for the intendedpurpose. Thus, for use as a therapeutic, the peptide, polypeptide, orprotein provided by this invention should have an affinity (K_(d)) ofbetween about 10⁻⁷ to about 10⁻¹⁵ M. More preferably the affinity is10⁻⁸ to about 10⁻¹² M. Most preferably, the affinity is 10⁻¹⁰ to about10⁻¹² M. For use as a reagent in a competitive binding assay to identifyother ligands, the amino acid sequence preferably has affinity for thereceptor of between about 10⁻⁵ to about 10⁻¹² M.

The present invention describes several different binding motifs, whichbind to active sites on IR or IGF-1R. The binding motifs are definedbased on the analysis of several different amino acid sequences andanalyzing the frequency that particular amino acids or types of aminoacids occur at a particular position of the amino acid sequence asdescribed in the related applications of Beasley et al. InternationalApplication PCT/US00/08528, filed Mar. 29, 2000, and Beasley et al.,U.S. application Ser. No. 09/538,038, filed Mar. 29, 2000.

Also included within the scope of this invention are amino acidsequences containing substitutions, additions, or deletions based on theteachings disclosed herein and which bind to IR or IGF-1R with the sameor altered affinity. For example, sequence tags (e.g., FLAG® tags) oramino acids, such as one or more lysines, can be added to the peptidesequences of the invention (e.g., at the N-terminal or C-terminal ends)as described in detail herein. Sequence tags can be used for peptidepurification or localization. Lysines can be used to increase peptidesolubility or to allow for biotinylation. Alternatively, amino acidresidues located at the carboxy and amino terminal regions of theconsensus motifs described below, which comprise sequence tags (e.g.,FLAG® tags), or which contain amino acid residues that are notassociated with a strong preference for a particular amino acid, mayoptionally be deleted providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) such as lysine whichpromote the stability or biotinylation of the amino acids sequences maybe deleted depending on the use of the sequence, as for example,expression of the sequence as part of a larger sequence which issoluble, or linked to a solid support.

Peptides that bind to IR or IGF-1R, and methods and kits for identifyingsuch peptides, have been disclosed by Beasley et al., InternationalApplication PCT/US00/08528 filed Mar. 29, 2000 and Beasley et al., U.S.application Ser. No. 09/538,038 filed Mar. 29, 2000, which areincorporated by reference in their entirety.

A. Consensus Motifs

The following motifs have been identified as conferring binding activityto IR and/or IGF-1R:

1. X₁X₂X₃X₄X₅ (Formula 1; Group 1; the A6 motif) wherein X₁, X₂, X₄ andX₅ are aromatic amino acids, preferably, phenylalanine or tyrosine. Mostpreferably, X₁ and X₅ are phenylalanine and X₂ is tyrosine. X₃ may beany small polar amino acid, but is preferably selected from asparticacid, glutamic acid, glycine, or serine, and is most preferably asparticacid or glutamic acid. X₄ is most preferably tryptophan, tyrosine, orphenylalanine and most preferably tryptophan. Particularly preferredembodiments of the A6 motif are FYDWF (SEQ ID NO:1554) and FYEWF (SEQ IDNO:1555). The A6 motif possesses agonist activity at IGF-1R, but agonistor antagonist activity at IR depending on the identity of amino acidsflanking A6. See FIG. 5A.

Amino acid sequences that comprise the A6 motif and possess agonistactivity at IR, include but are not limited to, D117/H2C:FHENFYDWFVRQVSKK (SEQ ID NO:1556); D117/H2 minus terminal lysines:FHENFYDWFVRQVS (SEQ ID NO:1557); RP9: GSLDESFYDWFERQLGKK (SEQ IDNO:1558); RP9 minus terminal lysines: GSLDESFYDWFERQLG (SEQ ID NO:1559);and S175: GRVDWLQRNANFYDWFVAELG (SEQ ID NO:1560). Preferred RP9sequences include GLADEDFYEWFERQLR (SEQ ID NO:1561), GLADELFYEWFDRQLS(SEQ ID NO:1562), GQLDEDFYEWFDRQLS (SEQ ID NO:1563), GQLDEDFYAWFDRQLS(SEQ ID NO:1564), GFMDESFYEWFERQLR (SEQ ID NO:1565), GFWDESFYAWFERQLR(SEQ ID NO:1566), GFMDESFYAWFERQLR (SEQ ID NO:1567), andGFWDESFYEWFERQLR (SEQ ID NO:1568). Non-limiting examples of Group 1(Formula 1; A6) amino acid sequences are shown in FIGS. 1A-1O.

2. X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃ (Formula 2, Group 3; the B6 motif) wherein X₆and X₇ are aromatic amino acids, preferably, phenylalanine or tyrosine.Most preferably, X₆ is phenylalanine and X₇ is tyrosine. X₈, X₉, X₁₁,and X₁₂ may be any amino acid. X₁₀ and X₁₃ are hydrophobic amino acids,preferably leucine, isoleucine, phenylalanine, tryptophan or methionine,but more preferably leucine or isoleucine. X₁₀ is most preferablyisoleucine for binding to IR and leucine for binding to IGF-1R. X₁₃ ismost preferably leucine. Amino acid sequences of Formula 2 may functionas an antagonist at the IGF-1R, or as an agonist at the IR. Preferredconsensus sequences of the Formula 2 motif are FYX₈X₉LX₁₁X₁₂L (SEQ IDNO:1569), FYX₈X₉IX₁₁X₁₂L (SEQ ID NO:1570), FYX₈AIX₁₁X₁₂L (SEQ IDNO:1571), and FYX₈YFX₁₁X₁₂L (SEQ ID NO:1572).

Another Formula 2 motif for use with this invention comprises FYX₈YFX₁₁X₁₂L (SEQ ID NO:1573) and is shown as Formula 2A (“NNRP”) below:X₁₁₅X₁₁₆X₁₁₇X₁₁₈FYX₈YFX₁₁X₁₂LX₁₁₉X₁₂₀X₁₂₁X₁₂₂ (SEQ ID NO:1574) whereinX₁₁₅-X₁₁₈ and X₁₁₈-X₁₂₂ may be any amino acid which allows for bindingto IR or IGF-1R. X₁₁₅ is preferably selected from the group consistingof tryptophan, glycine, aspartic acid, glutamic acid, and arginine.Aspartic acid, glutamic acid, glycine, and arginine are more preferred.Tryptophan is most preferred. The preference for tryptophan is based onits presence in clones at a frequency three to five fold higher thanthat expected over chance for a random substitution, whereas asparticacid, glutamic acid and arginine are present about two fold over thefrequency expected for random substitution.

X₁₁₆ preferably is an amino acid selected from the group consisting ofaspartic acid, histidine, glycine, and asparagine. X₁₁₇ and X₁₁₈ arepreferably glycine, aspartic acid, glutamic acid, asparagine, oralanine. More preferably X₁₁₇ is glycine, aspartic acid, glutamic acidand asparagine whereas X₁₁₈ is more preferably glycine, aspartic acid,glutamic acid or alanine. X₈ when present in the Formula 2A motif ispreferably arginine, glycine, glutamic acid, or serine. X₁₁ when presentin the Formula 2A motif is preferably glutamic acid, asparagine,glutamine, or tryptophan, but most preferably glutamic acid. X₁₂ whenpresent in the Formula 2A motif is preferably aspartic acid, glutamicacid, glycine, lysine or glutamine, but most preferably aspartic acid.X₁₁₉ is preferably glutamic acid, glycine, glutamine, aspartic acid oralanine, but most preferably glutamic acid. X₁₂₀ is preferably glutamicacid, aspartic acid, glycine or glutamine, but most preferably glutamicacid. X₁₂₁ is preferably tryptophan, tyrosine, glutamic acid,phenylalanine, histidine, or aspartic acid, but most preferablytryptophan or tyrosine. X₁₂₂ is preferably glutamic acid, aspartic acidor glycine; but most preferably glutamic acid. Preferred amino acidresidue are identified based on their frequency in clones over two foldover that expected for a random event, whereas the more preferredsequences occur about 3-5 times as frequently as expected.

In certain cases, Formula 1 and Formula 2 amino acid sequences may alsoinclude two cysteine residues, which may be positioned either outside orinside the motif sequence (e.g., X₁ X₂ X₃X₄ X₅ andX₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃), as described herein. The spacing between thecysteine residues preferably may vary from 3 amino acids, e.g., RP62(CDFYCALSRLSGQPRDRMPNYPGTS; SEQ ID NO:XX) up to 19 amino acids, e.g.,RP35 (DRDFCRFYERLTALVGGQVDGGWPC; SEQ ID NO:XX). Formula 1 and Formula 2peptides may exhibit varying size and cysteine positioning. For example,Formula 2 peptide RP6 (TFYSCLASLLTGTPQPNRGPWERCR; SEQ ID NO:XX) andderivatives, RP30-IGF, RP33-IGF, include two cysteine residues separatedby 18 amino acids. In contrast, Formula 1 peptide G33(GIISQSCPESFYDWFAGQVSDPWWCW; SEQ ID NO:XX) includes two cysteinesseparated by 17 amino acid residues. In certain Formula and Formula 2peptides, the position and spacing of the cysteine residues was found tobe highly preferred in these peptides as determined by calculations ofamino acid preferences from peptides obtained by biopanning of RP6 andG33 secondary libraries. Without wishing to be bound by theory, it ispossible that the cysteine pairs observed in Formula 1 and Formula 2amino acid sequences form cysteine loop structures.

3. X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁ (Formula 3, reverse B6, revB6), wherein X₁₄and X₁₇ are hydrophobic amino acids; X₁₄, X₁₇ are preferably leucine,isoleucine, and valine, but most preferably leucine; X₁₅, X₁₆, X₁₈ andX₁₉ may be any amino acid; X₂₀ is an aromatic amino acid, preferablytyrosine or histidine, but most preferably tyrosine; and X₂₁ is anaromatic amino acid, but preferably phenylalanine or tyrosine, and mostpreferably phenylalanine. For use as an IGF-1R binding ligand, anaromatic amino acid is strongly preferred at X₁₈.

4. X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁X₃₂X₃₃X₃₄X₃₅X₃₆X₃₇X₃₈X₃₉X₄₀ X₄₁(Formula 4; Group 7; the F8 motif) wherein X₂₂, X₂₅, X₂₆, X₂₈, X₂₉, X₃₀,X₃₃, X₃₄, X₃₅, X₃₆, X₃₇, X₃₈, X₄₀, and X₄₁ are any amino acid. X₃₅ andX₃₇ may be any amino acid when the F8 motif is used as an IR bindingligand or as a component of an IR binding ligand, however for use as anIGF-1R binding ligand, glycine is strongly preferred at X₃₇ and ahydrophobic amino acid, particularly, leucine, is preferred at X₃₅. X₂₃is a hydrophobic amino acid. Methionine, valine, leucine or isoleucineare preferred amino acids for X₂₃, however, leucine which is mostpreferred for preparation of an IGF-1R binding ligand is especiallypreferred for preparation of an IR binding ligand. At least one cysteineis located at X₂₄ through X₂₇, and one at X₃₉ or X₄₀. Together thecysteines are capable of forming a cysteine cross-link to create alooped amino acid sequence. In addition, although a spacing of 14 aminoacids in between the two cysteine residues is preferred, other spacingsmay also be used provided binding to IGF-1R or IR is maintained.Accordingly, other amino acids may be substituted for the cysteines atpositions X₂₄ and X₃₉ if the cysteines occupy other positions.

In one embodiment, for example, the cysteine at position X₂₄ may occurat position X₂₇ which will produce a smaller loop provided that thecysteine is maintained at position X₃₉. These smaller looped peptidesare described herein as Formula 5, infra. X₂₇ is any polar amino acid,but is preferably selected from glutamic acid, glutamine, aspartic acid,asparagine, or as discussed above cysteine. The presence of glutamicacid at position X₂₇ decreases binding to IR but has less of an effecton binding to IGF-1R. X₃₁ is any aromatic amino acid and X₃₂ is anysmall amino acid. For binding to IGF-1R, glycine or serine is preferredat position X₃₁, however, tryptophan is highly preferred for binding toIR. At position X₃₂, glycine is preferred for both IGF-1R and IRbinding. X₃₆ is an aromatic amino acid. A preferred consensus sequencefor F8 is X₂₂LCX₂₅X₂₆EX₂₈X₂₉X₃₀WGX₃₃X₃₄X₃₅ X₃₆X₃₇X₃₈CX₄₀X₄₁ (SEQ IDNO:1575) whereas the amino acids are defined above. A more preferred F8sequence is HLCVLEELFWGASLFGYCSG (“F8”; SEQ ID NO:1576). Amino acidsequences comprising the F8 sequence motif preferably bind to IR overIGF-1R. FIGS. 2A-2E list non-limiting examples of Formula 4 amino acidsequences.

5. X₄₂X₄₃X₄₄X₄₅X₄₆X₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃X₅₄X₅₅X₅₆X₅₇X₅₈X₅₉X₆₀X₆₁ (Formula5; mini F8 motif) wherein X₄₂, X₄₃, X₄₄, X₄₅, X₅₃, X₅₅, X₅₆, X₅₈, X₆₀,and X₆₁ are any amino acid. X₄₃, X₄₆, X₄₉, X₅₀, and X₅₄ are hydrophobicamino acids, however, X₄₃ and X₄₆ are preferably leucine, whereas X₅₀ ispreferably phenylalanine or tyrosine but most preferably phenylalanine.X₄₇ and X₅₉ are cysteines. X₄₈ is preferably a polar amino acid, i.e.,aspartic acid or glutamic acid, but most preferably glutamic acid. Useof the small amino acid at position 54 may confer IGF-1R specificity.X₅₁, X₅₂, and X₅₇ are small amino acids, preferably glycine. A preferredconsensus sequence for mini F8 is X₄₂X₄₃X₄₄X₄₅LCEX₄₉FGGX₅₃X₅₄X₅₅X₅₆GX₅₈CX₆₀X₆₁ (SEQ ID NO:1577). Amino acid sequences comprising thesequence of Formula 5 preferably bind to IGF-1R or IR.

6. X₆₂X₆₃X₆₄X₆₅X₆₆X₆₇X₆₈X₆₉X₇₀X₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇X₇₈X₇₉X₈₀ X₈₁(Formula 6; Group 2; the D8 motif) wherein X₆₂, X₆₅, X₆₈, X₆₉, X₇₁, X₇₃,X₇₆, X₇₇, X₇₈, X₈₀, and X₈₁ may be any amino acid. X₆₆ may also be anyamino acid, however, there is a strong preference for glutamic acid.Substitution of X₆₆ with glutamine or valine may result in attenuationof binding. X₆₃, X₇₀, and X₇₄ are hydrophobic amino acids. X₆₃ ispreferably leucine, isoleucine, methionine, or valine, but mostpreferably leucine. X₇₀ and X₇₄ are preferably valine, isoleucine,leucine, or methionine. X₇₄ is most preferably valine. X₆₄ is a polaramino acid, more preferably aspartic acid or glutamic acid, and mostpreferably glutamic acid. X₆₇ and X₇₅ are aromatic amino acids. Whereastryptophan is highly preferred at X₆₇, X₇₅ is preferably tyrosine ortryptophan but most preferably tyrosine. X₇₂ and X₇₉ are cysteines thatagain are believed to form a loop which position amino acid may bealtered by shifting the cysteines in the amino acid sequence.

D8 is most useful as an amino acid sequence having a preference forbinding to IR as only a few D8 sequences capable of binding to IGF-1Rover background have been detected. A preferred sequence for binding toIR is X₆₂LX₆₄X₆₅X₆₆WX₆₈X₆₉X₇₀X₇₁CX₇₃X₇₄X₇₅X₇₆X₇₇X₇₈CX₈₀X₈₁ (SEQ IDNO:1578). Examples of specific peptide sequences comprising this motifinclude D8 KWLDQEWAWVQCEVYGRGCPSKK (SEQ ID NO:1579); and D8 minusterminal lysines: KWLDQEWAWVQCEVYGRGCPS (SEQ ID NO:1580). Preferred D8monomer sequences include SLEEEWAQIQCEIYGRGCRY (SEQ ID NO:1581) andSLEEEWAQIQCEIWGRGCRY (SEQ ID NO:1582). Preferred D8 dimer sequencesinclude SLEEEWAQIECEVYGRGCPS (SEQ ID NO:1583), and SLEEEWAQIECEVWGRGCPS(SEQ ID NO:1584). Non-limiting examples of Group 2 (Formula 6; D8) aminoacid sequences are shown in FIGS. 3A-3E.

7. HX₈₂X₈₃X₈₄X₈₅X₈₆X₈₇X₈₈X₈₉X₉₀X₉₁X₉₂ (Formula 7) wherein X₈₂ is prolineor alanine but most preferably proline; X₈₃ is a small amino acid morepreferably proline, serine or threonine and most preferably proline; X₈₄is selected from leucine, serine or threonine but most preferablyleucine; X₈₅ is a polar amino acid preferably glutamic acid, serine,lysine or asparagine but more preferably serine; X₈₆ may be any aminoacid but is preferably a polar amino acid such as histidine, glutamicacid, aspartic acid, or glutamine; X₈₇ is an aliphatic amino acidpreferably leucine, methionine or isoleucine and most preferablyleucine; amino acid X₈₈, X₈₉ and X₉₀ may be any amino acids; X₉₁ is analiphatic amino acid with a strong preference for leucine as is X₉₂.Phenylalanine may also be used at position 92. A preferred consensussequence of Formula 7 is HPPLSX₈₆LX₈₈X₈₉X₉₀LL (SEQ ID NO:1585). TheFormula 7 motif binds to IR with little or no binding to IGF-1R.

8. Another sequence is X₁₀₄X₁₀₅X₁₀₆X₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁X₁₁₂X₁₁₃ X₁₁₄(Formula 8) which comprises eleven amino acids wherein at least one, andpreferably two of the amino acids of X₁₀₆ through X₁₁₁ are tryptophan.In addition, it is also preferred that when two tryptophan amino acidsare present in the sequence they are separated by three amino acids,which are preferably, in sequential order proline, threonine andtyrosine with proline being adjacent to the tryptophan at the aminoterminal end. Accordingly, the most preferred sequence forX₁₀₇X₁₀₈X₁₀₉X₁₁₀X₁₁₁ is WPTYW (SEQ ID NO:1586). At least one of thethree amino acids on the amino terminal (X₁₀₄, X₁₀₅, X₁₀₆) and at leastone of the amino acids carboxy terminal (X₁₁₂, X₁₁₃, X₁₁₄) endsimmediately flanking X₁₀₇-X₁₁₁ are preferably a cysteine residue, mostpreferably at X₁₀₅ and X₁₁₃ respectively. Without being bound by theory,the cysteines are preferably spaced so as to allow for the formation ofa loop structure. X₁₀₄ and X₁₁₄ are both small amino acids such as, forexample, alanine and glycine. Most preferably, X₁₀₄ is alanine and X₁₁₄is glycine. X₁₀₅ may be any amino acid but is preferably valine. X₁₁₂ ispreferably asparagine. Thus, the most preferred sequence is ACVWPTYWNCG(SEQ ID NO:1587).

9. An amino acid sequence comprising JBA5: DYKDLCQSWGVRIGWLAGLCPKK (SEQID NO:1541); or JBA5 without terminal lysines: LCQSWGVRIGWLAGLCP (SEQ IDNO:1542) (Formula 9). The Formula 9 motif is another motif believed toform a cysteine loop that possesses agonist activity at both IR andIGF-1R. Although IR binding is not detectable by ELISA, binding ofFormula 9 to IR is competed by insulin and is agonistic.

10. W X₁₂₃ G Y X₁₂₄ W X₁₂₅ X₁₂₆ (SEQ ID NO:1543) (Formula 10; Group 6)wherein X₁₂₃ is selected from proline, glycine, serine, arginine,alanine or leucine, but more preferably proline; X₁₂₄ is any amino acid,but preferably a charged or aromatic amino acid; X₁₂₅ is a hydrophobicamino acid preferably leucine or phenylalanine, and most preferablyleucine. X₁₂₆ is any amino acid, but preferably a small amino acid. Inone embodiment of the present invention, the Formula 10, Group 6 motifis WPGY (SEQ ID NO:1588). Examples of specific peptide sequencescomprising this motif include E8: KVRGFQGGTVWPGYEWLRNAAKK (SEQ IDNO:1589); and E8 minus terminal lysines: KVRGFQGGTVWPGYEWLRNAA (SEQ IDNO:1590). Preferred Group 6 sequences include WAGYEWF (SEQ ID NO:1591),WEGYEWL (SEQ ID NO:1592), WAGYEWL (SEQ ID NO:1593), WEGYEWF (SEQ IDNO:1594), and DSDWAGYEWFEEQLD (SEQ ID NO:1595). Non-limiting examples ofGroup 6 amino acid sequences are shown in FIGS. 4A-4B.

The IR and IGF-1R binding activities of representative Group 1 (Formula1; A6); Group 2 (Formula 6; D8); and Group 6 (Formula 10); and Group 7(Formula 4; F8) amino acid sequences are summarized in FIGS. 8 and9A-9B. Group 1 (Formula 1; A6) amino acid sequences contain theconsensus sequence FyxWF (SEQ ID NO:1596), which is typically agonisticin cell-based assays. Group 2 (Formula 6; D8) amino acid sequences arecomposed of two internal sequences having a consensus sequence VYGR (SEQID NO:1597) and two cysteine residues each. Thus, Group 2 peptides arecapable of forming a cyclic peptide bridged with a disulfide bond.Neither of these consensus sequences have any significant linearsequence similarities greater than 2 or 3 amino acids with matureinsulin. Group 7 (Formula 4; F8) amino acid sequences are composed oftwo internal exemplary sequences which do not have any significantsequence homology, but have two cysteine residues 13-14 residues apart,thus being capable of forming a cyclic peptide with a long loop anchoredby a disulfide bridge.

B. Amino and Carboxyl Terminal Extensions Modulate Activity of Motifs

In addition to the motifs stated above, the invention also providespreferred sequences at the amino terminal or carboxyl terminal endswhich are capable of enhancing binding of the motifs to either IR,IGF-1R, or both. In addition, the use of the extensions described belowdoes not preclude the possible use of the motifs with othersubstitutions, additions or deletions that allow for binding to IR,IGF-1R, or both.

1. Formula 1

Any amino acid sequence may be used for extensions of the amino terminalend of A6, although certain amino acids in amino terminal extensions maybe identified which modulate activity. Preferred carboxy terminalextensions for A6 are A6-X₉₃X₉₄X₉₅X₉₆X₉₇ wherein X₉₃ may be any aminoacid, but is preferably selected from the group consisting of alanine,valine, aspartic acid, glutamic acid, and arginine, and X₉₄ and X₉₇ areany amino acid; X₉₅ is preferably glutamine, glutamic acid, alanine orlysine but most preferably glutamine. The presence of glutamic acid atX₉₅ however may confer some IR selectivity. Further, the failure toobtain sequences having an asparagine or aspartic acid at position X₉₅may indicate that these amino acids should be avoided to maintain orenhance sufficient binding to IR and IGF-1R. X₉₆ is preferably ahydrophobic or aliphatic amino acid, more preferably leucine,isoleucine, valine, or tryptophan but most preferably leucine.Hydrophobic residues, especially tryptophan at X₉₆ may be used toenhance IR selectivity.

2. Formula 2

B6 with amino terminal and carboxy terminal extensions may berepresented as X₉₈X₉₉-B6-X₁₀₀. X₉₈ is optionally aspartic acid and X₉₉is independently an amino acid selected from the group consisting ofglycine, glutamine, and proline. The presence of an aspartic acid at X₉₈and a proline at X₉₉ is associated with an enhancement of binding forboth IR and IGF-1R. A hydrophobic amino acid is preferred for the aminoacid at X₁₀₀, an aliphatic amino acid is more preferred. Most preferablyleucine, for IR and valine for IGF-1R. Negatively charged amino acidsare preferred at both the amino and carboxy terminals of Formula 2A.

3. Formula 3

An amino terminal extension of Formula 3 defined as X₁₀₁X₁₀₂X₁₀₃-revB6wherein X₁₀₃ is a hydrophobic amino acid, preferably leucine, isoleucineor valine, and X₁₀₂ and X₁₀₁ are preferably polar amino acids, morepreferably aspartic acid or glutamic acid may be useful for enhancingbinding to IR and IGF-1R. No preference is apparent for the amino acidsat the carboxy terminal end of Formula 3.

4. Formula 10

In one preferred embodiment, Formula 10 sequences WX₁₂₃GYX₁₂₄WX₁₂₅X₁₂₆(SEQ ID NO:1543) can include an amino terminal extension comprising thesequence DSD and/or a carboxy terminal extension comprising the sequenceEQLD (SEQ ID NO:1598).

C. IR Binding Preferences

As indicated above, the amino acid sequences containing the motifs ofthis invention may be constructed to have enhanced selectivity foreither IR or IGF-1R by choosing appropriate amino acids at specificpositions of the motifs or the regions flanking them. By providing aminoacid preferences for IR or IGF-1R, this invention provides the means forconstructing amino acid sequences with minimized activity at thenon-cognate receptor. For example, the amino acid sequences disclosedherein with high affinity and activity for IR and low affinity andactivity for IGF-1R are desirable as IR agonist as their propensity topromote undesirable cell proliferation, an activity of IGF-1 agonists,is reduced. Ratios of IR binding affinity to IGF-1R binding affinity forspecific sequences are provided in FIGS. 1A-1O; 2A-2E; 3A-3E; 4A-4I;44A-44B. As an insulin therapeutic, the IR/IGF-1R binding affinity ratiois preferably greater than 100. Conversely, for use as an IGF-1Rtherapeutic, the IR/IGF-1R ratio should be less than 0.01. Examples ofpeptides that selectively bind to IGF-1R are shown below.

TABLE 1 IGF-IR-SELECTIVE SEQUENCES Ratios over Background ComparisonsSEQ ID Clone NO: Sequence E-Tag IGF-IR IR IGF-1R/IR IR/IGF-1R FORMULA 1(Group 1; A6-like): A6L-0-E6-IR 1599YRGMLVLGRSSDGAGKVAFERPARIGQTVFAVNFYDWFV 31.0 31.0 1.8 17.0 0.1H2CA-4-G9-IGFR 1600 GIISQSCPESFYDWPAGQVSDPWWCW 8.6 9.5 0.6 16.0 0.1H2CA-4-H6-IGFR 1601 VGRASGFPENFYDWFGRQLSLQSGEQ 4.9 10.5 0.7 14.6 0.1A6L-0-E4-IR 1602 YRGMLVLGRISDGAG#VASEPPARIGRKVFAVNFYDWFV 26.0 16.0 1.313.0 0.1 A6L-0-H3-IR 1603 YRGMLVLGRISGGAGKAASERPARIGQKVSAVNFYDWFV 27.026.0 2.0 13.0 0.1 H2CA-4-F5-IGFR 1604 VGYQGQGDENFYDWFIRQVSGRLGVQ 5.5 9.70.8 12.3 0.1 H2CA-4-H8-IGFR 1605 SACQPDCHENFYDWFARQVSGGAAYG 5.6 9.2 1.09.4 0.1 H2CA-4-F11-IGFR 1606 SAAQLFFQESFYDWFLRQVAESSQPN 3.5 6.8 1.0 6.70.1 H2CA-4-F6-IGFR 1607 AVRAIRFDEAFYDWFVRQISDGQGNK 3.9 7.3 1.1 6.4 0.2H2CA-4-P10-IGFR 1608 VNQSGSIHENFYDWFERQVSHQRGVR 4.9 5.7 1.0 5.9 0.2H2CA-1-A3-IGFR 1609 APDPSDFQEIFYDWFVRQVSRNPGGG 7.7 3.8 0.8 5.1 0.2H2CA-3-C8-IGFR 1610 SSCDGAGHESFYEYNFVRQVSGCRSV 15.1 5.6 1.2 4.8 0.2H2CA-2-B9-IGFR 1611 RAGSSDFHEDFYEWFVRQVSLSLKGK 9.3 7.0 1.7 4.2 0.2H2CA-4-H4-IGFR 1612 QAVQPGFHEEFYDYNFVRQVSTGVGGG 3.9 4.1 1.0 4.2 0.2E4Dα-4-H2-IR 1613 GFREGNFYEWFQAQVT 37.8 33.9 8.2 4.1 0.2 H2CA-4-F7-IGFR1614 SSIGGGFHENFYDWFSRQLSQSPPLK 1.5 3.2 0.8 4.1 0.2 H2CA-3-D6-IGFR 1615QSPVGSSHEDFYDWFPRQVAQSGAHQ 8.3 9.0 2.2 4.0 0.3 H2CA-3-D8-IGFR 1616NYRRQVPNGNFYDWFDRQVFSLVTPG 10.9 7.2 1.8 4.0 0.3 H2CA-4-G11-IGFR 1617TLDGGSFEEQFYDWFVRQLSYRTNPD 10.8 9.5 2.5 3.9 0.3 H2CA-4-P1-IGFR 1618FYVQQYWGHENFYDWFDRQVSQSGGAG 5.8 3.5 0.9 3.8 0.3 H2CA-3-D7-IGFR 1619LRRQAPVEENFYDWFVRQVSGDRVGG 13.3 3.0 0.8 3.7 0.3 H2CA-1-A7-IGFR 1620RCGRELYHSTFYDWFDRQVAGRTCPS 8.0 2.2 0.6 3.7 0.3 H2CA-2-B4-IGFR 1621CCLLCRFQQNFYDWFVCQGISRLRPL 3.5 4.1 1.1 3.6 0.3 H2CA-2-B3-IGFR 1622PPLASDLDVQFYGWFVQQVSPPGRGG 7.7 3.8 1.0 3.6 0.3 H2CA-2-B2-IGFR 1623GAPVDQLHEDFYDWFVRQVSQAATG 4.1 3.4 1.0 3.5 0.3 E4Dα-2-D11-IGFR 1624GFREGSFYDWFQAQVT 40.2 11.1 3.3 3.4 0.3 20E2Bβ-4-G6-IR 1625SQAGSAFYAWFDQVLRTVHSA 22.4 6.2 1.9 3.3 0.3 H2CA-4-H9-IGFR 1626RGAVAGPHDQFYDWFDRQVSRVHKPG 8.7 5.6 1.9 3.0 0.3 H2CA-2-B11-IGFR 1627AICDAGFHEHFYDWFALQVSDCGRQS 11.9 4.6 1.6 3.0 0.3 H2CA-3-E8-IGFR 1628LGYQEPFQQNFYDWFVRQVSGAENAG 13.2 6.3 2.2 2.9 0.3 A6S-2-D11-IR 1629EAASLGSQDRNFYDWFVRQVV 48.4 37.4 13.5 2.8 0.4 A6S-2-D1-IR 1630VERSASSQDGNFYDWFVVQIR 37.8 30.6 12.0 2.6 0.4 A6S-3-E2-IR 1631TSEVQRRSQDNGYDWFVAQVA 33.1 24.7 9.8 2.5 0.4 H2CA-3-E11-IGFR 1632HLADGQFHEKFYDWFERQISSRCNDC 4.7 2.2 1.0 2.2 0.5 0 H2CA-3-C11-IGFR 1633FRTLAAQHDSFYDWFDRQVSGAAGER 9.3 3.3 1.6 2.1 0.5 A6-PD1-IGFR 1634SFHEDFYDWFDRQVSGSLKK H2C-PD1-IGFR(RP9) 1558 GSLDESFYDWFERQLGKK FORMULA 2(Group 2; B6-like) 20C-3-G3-IGFR 1635 TFYSCLASLLTGTPQPNRGPWERCE 33.132.3 1.2 27.0 <0.1 20C-4-C7-IGFR 1636 FFYDCLAALLQGVARYHDLCAVEIT 35.328.0 1.3 21.8 <0.1 B6Hα-1-B5-IR 1637 CCTTEMVVMDARDDPFYHKLSELVTGG 41.520.5 1.0 20.5 0.0 R20β-4-A6-IR 1638 RGQSDAFYSGLWALIGLSDG 9.3 25.9 1.517.3 0.1 20E2B-1-A6-IGFR 1639 GVRAMSFYDALVSVLGLGPSG 18.6 18.1 1.1 16.80.1 R20α-4-20A12-IR 1640 RLFYCGIQALGANLGYSGCV 48.6 39.9 2.4 16.6 0.120E2Bβ-4-G7-IR 1641 LQPCSGFYECIERLIGVKLSG 19.9 25.2 1.6 15.8 0.1NNRPγ-4-B11-IR 1642 LKDGFYDYFWQRLHLGS 4.1 18.7 1.2 15.5 0.120E2B-3-C6-IGFR 1643 VEGRGLFYDLLRQLLARRQNG 17.9 16.8 1.1 14.8 0.1B6Hα-1-A2-IR 1644 RGCNDDGGKGWSDDPFYHKLSELICGG 22.3 14.6 1.0 14.6 0.120E2A-4-F11-IGFR 1645 QGGSASFYDAIDRLLRMRIGG 21.3 18.8 1.3 14.6 0.1B6Hα-3-E9-IR 1646 RCEEKQAEVGPSSDPFYHKMSELLGCR 44.6 24.2 1.7 14.2 0.120C-3-F6-IGFR 1647 DRDFCRFYRLTALVGGQVDGGWPC 33.5 26.1 1.9 14.1 0.120E2B-4-H3-IGFR 1648 KLHNLMFYYGLQRLVWGAGLG 11.2 14.8 1.1 13.9 0.120E2B-3-C2-IGFR 1649 GNGDGMFYQLLSLLVGRDMHV 13.1 8.9 0.6 13.8 0.120C-3-A1-IGFR 1650 SSYGCDFYLMLFSLGLVASQELEC 26.5 20.8 1.5 13.7 0.120E2B-3-E3-IGFR 1651 PDLHKGFYAQLAQLIRGQLLS 22.4 16.3 1.3 13.1 0.1R20α-3-20E2-IR 1652 FYDAIDQLVRGSARAGGTRD 46.3 39.9 3.1 12.9 0.120E2B-4-H12-IGFR 1653 YSCGDGFYSLLSDLLGGQFRC 6.5 9.7 0.8 12.8 0.1B6Hα-3-F11-IR 1654 RGMKEEVLVGGSTDPFYHKLSELLQGS 49.5 18.7 1.6 11.7 0.120E2B-3-D2-IGFR 1655 IQQELTFYDLLHRLVRSELGS 20.7 12.4 1.1 11.7 0.120E2B-3-D8-IGFR 1656 GGTEVDFYRALERLVRGQLGL 20.4 17.7 1.6 11.3 0.120E2B-3-E8-IGFR 1657 LRIANLFYQRLWDLAFGGGG 15.7 16.7 1.5 11.1 0.1B6Hα-2-C4-IR 1658 RCGRW*AEMGAGDDPFYHKLSELVCG 20.7 9.9 0.9 11.0 0.1R2Dα-4-20C11-IR 1659 DRAFYNGLRDLVGAVYGAWD 43.7 30.8 3.0 10.3 0.120E2B-4-F8-IGFR 1660 PVGVQGFYEGLSRLVLGRGGW 12.3 7.3 0.8 9.7 0.120E2B-1-A11-IGFR 1661 RFSTDGFYQYLLALVGGGPVG 15.0 9.5 1.0 9.7 0.120E2B-3-D4-IGFR 1662 NSRDGGFYLQLERLLGFPVTG 8.1 7.9 0.8 9.6 0.120E2B-2-B11-IGFR 1663 VVTPVNGRRALEALVRG.RLG 13.9 10.6 1.1 9.4 0.120E2B-3-C8-IGFR 1664 QPAPDGFYSALMKLIGRGGVS 18.5 15.6 1.8 8.9 0.120E2B-2-B2-IGFR 1665 PGTDLGFYQALRCVVIQGACD 11.7 4.9 0.6 8.1 0.120E2B-4-F10-IGFR 1666 AQPCGGFYGLLEQLVGRSVCD 19.0 17.3 2.2 7.8 0.120E2B-4-F9-IGFR 1667 QPDHSYFYSLLQELVGSEERL 11.9 14.7 1.9 7.7 0.120C-30A4-IGFR 1668 QFYGCLLDLSLGVPSFGWRRRCITA 17.7 8.8 1.2 7.6 0.120E2B-3-D11-IGFR 1669 LGVTDGFYAALGYLIHGVGQF 14.3 12.2 1.6 7.6 0.120E2B-3-C11-IGFR 1670 CMM.DGFYAGLGCLLTAGEGR 15.3 15.4 2.1 7.5 0.120E2B-2-B3-IGFR 1671 ICTGQGFYQVLCGLLRGTSAR 9.1 5.3 0.7 7.4 0.120E2B-3-D12-IGFR 1672 QGNVLDFYGWIGRLLAKQGSD 10.3 6.2 0.9 7.3 0.120E2B-3-E21-IGFR 1673 VATSQGFYSGLSELLQGGGNV 13.9 6.0 0.8 7.3 0.120E2B-2-B8-IGFR 1674 IWATGDFYRLLSQLVMGRVFT 17.4 5.7 0.8 7.2 0.1NNRPγ-4-A9-IR 1675 EGSGFYGYFFSLLGLQG 3.0 10.0 1.4 7.1 0.120E2B-4-G11-IGFR 1676 RQGTGSFYLMLEQLLVGARGP 8.9 4.5 0.6 7.0 0.120E2B-3-D6-IGFR 1677 DSVGDNFYQLLESLVGGHGVG 20.7 17.8 2.6 6.9 0.1B6Hα-2-C7-IR 1678 RGIVAMVEATEVGSDHDPFYHKLSELVQGS 45.1 6.7 1.0 6.7 0.120E2B-2-B7-IGFR 1679 LSSDGQFYRALNLLLQGSAGR 18.0 6.1 0.9 6.7 0.120E2B-3-C4-IGFR 1680 ASSASGFYELLQRLAGLGLEV 23.4 20.4 3.3 6.2 0.220C-3-E4-IGFR 1681 FFYRCLSRLLGGQLGSRLGLSCIGD 37.7 7.7 1.3 6.0 0.2NNRPγ-4-A1-IR 1682 IIGGFYSYFNSVLRLGT 9.7 10.9 1.8 6.0 0.220E2B-4-H8-IGFR 1683 PAGPCGFYCGLGLLLHGDQSP 7.2 5.3 0.9 5.9 0.220E2B-4-H9-IGFR 1684 RCQGRGFYTCIQELIGFGDPD 4.5 5.2 0.9 5.6 0.2B6Hα-2-C10-IR 1685 SGAKVIVVTGDSGDPFYHKLSELLQGS 46.9 5.8 1.1 5.3 0.220E2A-3-C7-IGFR 1686 VGTVAGFYDAIAQLVARASRV 17.6 5.4 1.1 5.1 0.220E2B-I-A8-IGFR 1687 TLRSPTFYDWLEMVLTHGQGG 16.1 4.4 0.9 5.0 0.2NNRPγ-4-A7-IR 1688 RFDPFYSYFVNLLGASA 2.5 6.3 1.3 4.9 0.2 B6Hα-3-E8-IR1689 RGKTAVVIVGRPADPFYHKLSELLQGG 47.6 5.3 1.1 4.8 0.2 B6Hα-3-F10-IR 1690GCVVEWQKWHGASDPFYHKLSELGGCS 47.2 8.8 1.9 4.6 0.2 B6Hα-2-D6-IR 1691GRTMAVMAAGGPDDPFYHKLSELVQGG 33.5 4.4 1.0 4.4 0.2 B6Hα-3-E7-IR 1692GCAVVEEAERSRGDPFYHKLSELIQGC 47.0 5.6 1.3 4.3 0.2 B6Hα-2-D1-IR 1693GCEVIVEEGDSADPFYHKLSELCQGS 11.7 5.4 1.3 4.2 0.2 20E2A-3-D10-IGFR 1694MMVVDGFYDALHQLVVAQSLG 20.6 6.9 1.8 3.9 0.3 20E2A-3-A12-IGFR 1695LSVALSFYDALGQLVAGEGRW 16.1 4.3 1.1 3.9 0.3 B6Hα-4-GB-IR 1696GGTKAVAKVGTRDDPFYHKLSELLQGS 32.3 6.1 1.7 3.6 0.3 B6L-4-D7-IR 1697AETSVQVGWIRLQSVWPGEHWNTVDPFYHKLSELLRGSGA 14.3 4.8 1.4 3.4 0.3B6Hα-1-A3-IR 1698 SRAKVEAEMPDSGDPFYHKLSELLASG 37.4 2.6 0.8 3.3 0.3B6Hα-3-F7-IR 1699 SRVAATKEKRPSDDPFYHKLSELLQGS 41.5 3.1 1.0 3.1 0.3B6Hα-2-D8-IR 1700 SSETAKMVTGTRDDPFYHKLSELVQGS 19.3 3.0 1.0 3.0 0.3B6Hα-1-B3-IR 1701 GCITAENGAGDPFYHKLSELGGCS 33.1 3.2 1.1 2.9 0.3B6Hα-3-E5-IR 1702 RCGDEEGWQENRRDDPFYHKLSELFGGC 28.8 2.9 1.0 2.9 0.320E21-4-G11-IGFR 1703 MNVFVSFYDAIDQLVCQRIGC 20.7 3.3 1.3 2.6 0.420E2β-3-C7-IR 1704 QSGSGDFYDWLSRLIRGNGDG 1.5 3.1 1.5 2.0 0.5B6Hα-3-E6-IR 1705 CGAKMTGTPNDPFYHKLSELLQRG 18.2 2.3 1.2 1.9 0.520E2A-3-A3-IGFR 1706 GHYFGSFYDAIDQLVAGMLPG 5.2 3.0 1.5 1.9 0.5B6L-4-A7-IR 1707 AGTPAQVG*NRLWSVWPGEHWNTVDPFYNKLSELLRESGA 11.6 3.4 1.91.8 0.6 B6Hα-3-F1-IR 1708 CSMAAVAEAGDDDDPFYHKLSELCQGS 22.5 2.4 1.3 1.80.5 B6L-3-G6-IR 1709 VDTPAQVGWNRLWSVGPGEHWYTDDPFYH*LSELLRESGA 7.6 2.51.8 1.4 0.7 B6L-3-G5-IR 1710 AETSAQVGWQRLWSVWPGDHWSTLDPFYHKLSELLRESGA11.5 2.0 1.4 1.4 0.7 20B2A-3-A4-IGFR 1711 AGSVTSFYDAMEQLVATGTSA 16.8 2.51.8 1.4 0.7 B6-PD1-IGFR 1712 TDDGFYDALEQLVQGSKK 20E2-PD1-IGFR(RP10) 1713GSFYEALQRLVGGEQGKK FORMULA 10 (Group 6): R20-β-4-E8-IR 1714VRGFQGGTVWPGYEWLRNAA 41.0 34.9 3.6 9.7 0.1 40F-4-D1-IGFR 1715LSCLAYSRHGIWRPSTDLGLGRSVGEGSVSTRWRGYDWFE 4.9 4.6 0.3 13.1 0.140F-4-B1-IGFR 1716 GLDHSDAVGVHLGFAWPAQARGRWEAGGLEDTWAGYDWL 4.1 3.0 0.213.1 0.1 40F-4-D1-IGFR 1717 W.GYAWL 4.9 4.5 0.4 11.7 0.1

Besides relative binding at IR or IGF-1R, relative efficacy at thecognate receptor is another important consideration for choosing apotential therapeutic. Thus, a sequence that is efficacious at IR buthas little or no significant activity at IGF-1R may also be consideredas an important IR therapeutic, irrespective of the relative bindingaffinities at IR and IGF-1R. For example, A6 selectivity for IR may beenhanced by including glutamic acid in a carboxyl terminal extension atposition X₉₅. IR selectivity of the B6 motif may be enhanced by having atryptophan or phenylalanine at X₁₁. Tryptophan at X₁₃ also favorsselectivity of IR. A tryptophan amino acid at X₁₃ rather than leucine atthat position also may be used to enhance selectivity for IR. In thereverse B6 motif, a large amino acid at X₁₅ favors IR selectivity.Conversely, small amino acids may confer specificity for IGF-1R. In theF8 motif, an L in position X₂₃ is essentially required for IR binding.In addition, tryptophan at X₃₁ is also highly preferred. At X₃₂, glycineis preferred for IR selectivity.

D. Multiple Binding Sites on IR and IGF-1R

The competition data disclosed herein reveals that at least two separatebinding sites are present on IR and IGF-1R which recognize the differentsequence motifs provided by this invention.

As shown in FIG. 6, competition data indicate that peptides comprisingthe A6 motifs compete for binding to the same site on IR (Site 1)whereas the D8 motifs compete for a second site (Site 2). Theidentification of peptides that bind to separate binding sites on IR andIGF-1R provides for various schemes of binding to IR or IGF-1R toincrease or decrease its activity. Examples of such schemes for IR areillustrated in FIG. 7.

The table below shows sequences based on their groups, which bind toSite 1 or Site 2.

TABLE 2 SEQ Clone Sequence ID NO: REPRESENTATIVE SITE 1 PEPTIDES A6-like(FYxWF) (SEQ ID NO:1596): G3 KRGGGTFYEWFESALRKHGAGKK 1718 H2VTFTSAVFHENFYDWFVRQVSKK 1719 H2C FHENFYDWFVRQVSKK 1556 A6S-IR3-E12GRVDWLQRNANFYDWFVAELG 1560 A6S-IR4-G1 NGVERAGTGDNFYDWFVAQLH 1720H2C8-R3-812 QSDSGTVHDRFYGWFRDTWAS 1721 20E2A-R3-B11GRFYGWFQDAIDQLMPWGFDP 1722 rB6-F6 RYGRWGLAQQFYDWFDR 1723 E4Dα-1-B8-IR~GFREGQRWYWFVAQVT 1724 H2CA-4-F11-IR TYKARFLHENFYDWFNRQVSQYFGRV 1725H2CB-R3-D2 WTDVDGFHSGFYRWFQNQWER 1726 H2CB-R3-D12 VASGHVLHGQFYRWFVDQFAL1727 H2CB-R4-H5 QARVGNVHQQFYEWFREVMQG 1728 H2C-B-E8*TGHRLGLDEQFYWWFRDALSG 1729 H2CB-3-B6-TR~ VGDFCVSHDCFYGWFLRESMQ 1730A65-iR2-C1 RMYFSTGAPQNFYDWFVQEWD 1731 B6-like (FYxxLxxL) (SEQ IDNO:1732): 20C11 KDRAFYNGLRDLVGAVYGAWDKK 1733 20E2DYKDFYDAIDQLVRGSARAGGTRDKK 1734 B62-R3-C7 EHWNTVDPFYFTLFEWLRESG 1735B62-R3-C10 EHWNTVDPFYQYFSELLRESG 1736 20E2B-3-B3-IRAGVNAGFYRYFSTLLDWWDQG 1737 20E2-B-E3* TQGWEPFYGWFDDVVAQMFEE 173820E2A-R4-F9 PPWGARFYDAIEQLVFDNLCC 1739 RPNN-4-G6-HOLO* RWFNFYGYFESLLTHFS1740 RPNN-4-F3-HOLO* HYNAFYEYFQVLLAETW 1741 20E2A-R4-E2IGRVRSFYDAIDKLFQSDWER 1742 RPNN-2-C1-IR* EGWDFYSYFSGLLASVT 174320E2B-4-F12-IR SVKEVQFYRYFYDLLQSEESG 1744 20E2-B-E12GNSGGSFYRYFQLLLDSDGMS 1745 20E2A-R3-B6 RDAGSSFYDAIDQLVCLTYFC 1746Reverse B6-like (LxxLxxYF) (SEQ ID NO:1747): r86-A12 LDALDRLMRYFEERPSL1748 r86-F9 PLAELWAYFEHSEQGRSSAH 1749 rB6-4-E7-IR LDPLDALLQYFWSVPGH 1750rB6-4-F9-IR RGRLGSLSTQFYNWFAE 1751 rB6-E6 ADELEWLLDYFMHQPRP 1752rB6-4-F12-IR DGVLEELFSYFSATVGP 1753 Group 6 (WFxYxWL) (SEQ ID NO:1754):R20β-4-A4-IR WPGLFFEEALQDWRGSTED 1755 Peptides by design**: H2C-PD1-IR~AAVHEQFYDWFADQYKK 1756 A6S-PD1-IR~ QAPSNFYDWFVREWDKK 1757 20E2-PD1-IR~QSFYDYIEELLGGEWKK 1758 B6C-PD1-IR~ DPFYQGLWEWLRESGKK 1759 REPRESENTATIVESITE 2 PEPTIDES (C-C LOOPS) F8-derived (Long C-C loop): F8HLCVLEELFWGASLFGYCSG 1760 F8-C12 FQSLLEELVWGAPLFRYGTG 1761 F8-Des2PLCVLEELFWGASLFGYCSG 1762 F8-F12 PLCVLEELFWGASLFGQCSG 1763 F8-B9HLCVLEELFWGASLFGQCSG 1764 F8-B12 DLRVLCELFGGAYVLGYCSE 1765 NNKH-2B3HRSVLKQLSWGASLFGQWAG 1766 NNKH-2F9~ HLSVGEELSWWVALLGQWAR 1767 NNKH-4H4~APVSTEELRWGALLFGQWAG 1768 D8-derived (Small C-C loop): D8KWLDQEWAWVQCEVYGRGCPSKK 1769 D8-G1 QLEEEWAGVQCEVYGRECPS 1770 D8-B5~ALEEEWAWVQVRSIRSGLPL 1771 D8-A7 SLDQEWAWVQCEVYGRGCLS 1772 D8-F1~WLEHEWAQTQCELYGRGCTY 1773 Midi C-C loop: D8-F10 GLEQGCPWVGLEVQCRGCPS1774 F8-B12~ DLRVLCELFGGAYVLGYCSE 1775 F8-A9 PLWGLCELFGGASLFGYCSS 1776**Based on analysis of entire panning data, amino acid preferences ateach position were calculated to define these “idealized” peptides;*Peptides synthesized and currently being purified; ˜Peptides planned.

In various aspects of the present invention, amino acid sequencescomprising Site 1 motifs may bind to Site 1 of IR or Site 1 of IGF-1R.Similarly, amino acids sequences comprising Site 2 motifs may bind toSite 2 of IR or Site 2 of IGF-1R. However, specific peptides may showhigher binding affinity for IR than for IGF-1R, while other peptides mayshow higher binding affinity for IGF-1R than for IR. In addition, Site 1and Site 2 on IR do not cross-talk, i.e., Site 1-binding sequences donot compete with Site 2-binding sequences at IR. In contrast, Site 1 andSite 2 on IGF-1R do show some cross-talk, suggesting an allostericeffect. These aspects are illustrated in the Examples describedhereinbelow.

E. Multivalent Ligands

This invention provides ligands that preferentially bind different siteson IR and IGF-1R. The A6 amino acid sequence motif confers binding to IRat Site 1 (FIG. 6). The D8 amino acid sequence motif confers binding toIR at Site 2 (FIG. 6). Accordingly, multimeric ligands may be preparedaccording to the invention by covalently linking amino acid sequences.Depending on the purpose intended for the multivalent ligand, amino acidsequences that bind the same or different sites may be combined to forma single molecule. Where the multivalent ligand is constructed to bindto the same corresponding site on different receptors, or differentsubunits of a receptor, the amino acid sequences of the ligand forbinding to the receptors may be the same or different, provided that ifdifferent amino acid sequences are used, they both bind to the samesite.

Multivalent ligands may be prepared by either expressing amino acidsequences which bind to the individual sites separately and thencovalently linking them together, or by expressing the multivalentligand as a single amino acid sequence which comprises within it thecombination of specific amino acid sequences for binding.

Various combinations of amino acid sequences may be combined to producemultivalent ligands having specific desirable properties. Thus, agonistsmay be combined with agonists, antagonists combined with antagonists,and agonists combined with antagonists. Combining amino acid sequencesthat bind to the same site to form a multivalent ligand may be useful toproduce molecules that are capable of cross-linking together multiplereceptor units. Multivalent ligands may also be constructed to combineamino acid sequences which bind to different sites (FIG. 7).

In view of the discovery disclosed herein of monomers having agonistproperties at IR or IGF-1R, preparation of multivalent ligands may beuseful to prepare ligands having more desirable pharmacokineticproperties due to the presence of multiple bind sites on a singlemolecule. In addition, combining amino acid sequences that bind todifferent sites with different affinities provides a means formodulating the overall potency and affinity of the ligand for IR orIGF-1R.

1. Construction of Hybrids

In one embodiment, hybrids of at least two peptides (e.g., dimerpeptides) may be produced as recombinant fusion polypeptides, which areexpressed in any suitable expression system. The polypeptides may bindthe receptor as either fusion constructs containing amino acid sequencesbesides the ligand binding sequences or as cleaved proteins from whichsignal sequences or other sequences unrelated to ligand binding areremoved. Sequences for facilitating purification of the fusion proteinmay also be expressed as part of the construct. Such sequencesoptionally may be subsequently removed to produce the mature bindingligand. Recombinant expression also provides means for producing largequantities of ligand. In addition, recombinant expression may be used toexpress different combinations of amino acid sequences and to vary theorientation of their combination, i.e., amino to carboxyl terminalorientation.

In one embodiment shown below (FIG. 28), MBP-FLAG®-PEPTIDE-(GGS)_(n)(SEQ ID NO:1777)-PEPTIDE-E-TAG, a fusion construct producing a peptidedimer comprises a maltose binding protein amino acid sequence (MBP) orsimilar sequence useful for enabling the affinity chromatographypurification of the expressed peptide sequences. This purificationfacilitating sequence may then be attached to a FLAG® sequence toprovide a cleavage site to remove the initial sequence. The dimer thenfollows which includes the intervening linker and a tag sequence may beincluded at the carboxyl terminal portion to facilitateidentification/purification of the expression of peptide. In therepresentative construct illustrated above, G and S are glycine andserine residues, which make up the linker sequence. As non-limitingexamples, n can be 1, 2, 3, or 4 to yield a linker sequence of 3, 6, 9,and 12 amino acids, respectively.

In addition to producing the dimer peptides by recombinant proteinexpression, dimer peptides may also be produced by peptide synthesiswhereby a synthetic technique such as Merrifield synthesis (Merrifield,1997), may be used to construct the entire peptide.

Other methods of constructing dimer peptides include introducing alinker molecule that activates the terminal end of a peptide so that itcan covalently bind to a second peptide. Examples of such linkersinclude, but are not limited to, diaminoproprionic acid activated withan oxyamino function. A preferred linker is a dialdehyde having theformula O═CH—(CH₂)_(n)—CH═O, wherein n is at least 2 to 6, but ispreferably 6 to produce a linker of about 25 to 30 angstroms in length.Other preferred linkers are shown in Table 3. Linkers may be used, forexample, to couple monomers at either the carboxyl terminal or the aminoterminal ends to form dimer peptides. Also, the chemistry can beinverted, i.e., the peptides to be coupled can be equipped with aldehydefunctions, either by oxidation with sodium periodate of an N-terminalserine, or by oxidation of any other vicinal hydroxy- or amino-groups,and the linker can comprise two oxyamino functions (e.g., at end of apolyethylene glycol linker) or amino groups which are coupled byreductive amination.

In specific embodiments, Site 1-Site 2 and Site 2-Site 1 orientationsare possible. In addition, N-terminal to N-terminal (N-N); C-terminal toC-terminal (C-C); N-terminal to C-terminal (N-C); and C-terminal toN-terminal (C-N) linkages are possible. Accordingly, peptides may beoriented Site 1 to Site 2, or Site 2 to Site 1, and may be linkedN-terminus to N-terminus, C-terminus to C-terminus, N-terminus toC-terminus, or C-terminus to N-terminus. In certain cases, a specificorientation may be preferable to others, for example, for maximalagonist or antagonist activity.

In an unexpected and surprising result, the orientation and linkage ofthe monomer subunits has been found to dramatically alter dimer activity(see Examples, below). In particular, certain Site 1/Site 2 heterodimersequences show agonist or antagonist activity at IR, depending onorientation and linkage of the constituent monomer subunits. Forexample, a Site 1-Site 2 orientation (C-N linkage), e.g., the S453heterodimer, shows antagonist activity at IR (FIG. 18A; Table 7). Incontrast, a Site 2-Site 1 orientation (C-N linkage), e.g., the S455heterodimer, shows potent agonist activity at IR (FIG. 18D; Table 7).Similarly, Site 1-Site 2 (C-N linkage) heterodimers, e.g., S425 andS459, show antagonist activity at IR (Table 7), while Site 1-Site 2 (C-Cor N-N linkage) heterodimers, e.g., S432-S438, S454, and S456, showagonist activity (Table 7).

Whether produced by recombinant gene expression or by conventionalchemical linkage technology, the various amino acid sequences may becoupled through linkers of various lengths. Where linked sequences areexpressed recombinantly, and based on an average amino acid length ofabout 4 angstroms, the linkers for connecting the two amino acidsequences would typically range from about 3 to about 12 amino acidscorresponding to from about 12 to about 48 Å. Accordingly, the preferreddistance between binding sequences is from about 2 to about 50 Å. Morepreferred is 4 to about 40. The degree of flexibility of the linkerbetween the amino acid sequences may be modulated by the choice of aminoacids used to construct the linker. The combination of glycine andserine is useful for producing a flexible, relatively unrestrictivelinker. A more rigid linker may be constructed by using amino acids withmore complex side chains within the linkage sequence.

2. Characterization of Specific Dimers

Specific dimers which are comprised of monomer subunits that both bindwith high affinity to the same site on IR or IGF-1R (e.g., Site 1-Site 1or Site 2-Site 2), or monomer subunits that bind to different sites onIR or IGF-1R (e.g., Site 1-Site 2 or Site 2-Site 1) are disclosedherein.

Other combinations of peptides are within the scope of this inventionand may be determined as demonstrated in the examples described herein.

F. Peptide Synthesis

Many conventional techniques in molecular biology, protein biochemistry,and immunology may be used to produce the amino acid sequences for usewith this invention. The present invention encompasses the specificamino acid sequences shown in FIGS. 1-4, 8, and 9 and Table 7, intera/ia, without additions (e.g., linker or spacer sequences) deletions,alterations, or modification. The present invention further encompassesvariants that include additional sequences, altered sequences, andfunctional fragments thereof. In a preferred embodiment, the amino acidsequence variant or fragment shares at least one function characteristic(e.g., binding, agonist, or antagonist activity) of the referencesequence. Variant peptides include, for example, genetically engineeredmutants, and may differ from the amino acid sequences shown in thefigures and tables of the application by the addition, deletion, orsubstitution of one or more amino acid residues. Alterations may occurat the amino- or carboxy-terminal positions of the reference amino acidsequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Inaddition, variants may comprise synthetic or non-naturally occurringamino acids in accordance with this invention.

Variant amino acid sequences can have conservative changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. More infrequently, avariant peptide can have non-conservative changes, e.g., substitution ofa glycine with a tryptophan. Guidance in determining which amino acidresidues can be substituted, inserted, or deleted without abolishingbinding or biological activity can be found using computer programswell-known in the art, for example, DNASTAR software (DNASTAR, Inc.,Madison, Wis.). Guidance is also provided by the data disclosed herein.In particular, FIGS. 1-4, 8, 9, 43, 44, and Table 7, inter alia, teachwhich amino acid residues can be deleted, added, substituted, ormodified, while maintaining the IR- or IGF-1R-related function(s) (e.g.,binding, agonist, or antagonist activity) of the amino acid sequences.

For the purposes of this invention, the amino acids are grouped asfollows: amino acids possessing alcohol groups are serine (S) andthreonine (T). Aliphatic amino acids are isoleucine (I), leucine (L),valine (V), and methionine (M). Aromatic amino acids are phenylalanine(F), histidine (H), tryptophan (W), and tyrosine (Y). Hydrophobic aminoacids are alanine (A), cysteine (C), phenylalanine (F), glycine (G),histidine (H), isoleucine (I), leucine (L), methionine (M), arginine(R), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).Negative amino acids are aspartic acid (D) and glutamic acid (E). Thefollowing amino acids are polar amino acids: cysteine (C), aspartic acid(D), glutamic acid (E), histidine (H), lysine (K), asparagine (N),glutamine (Q), arginine (R), serine (S), and threonine (T). Positiveamino acids are histidine (H), lysine (K), and arginine (R). Small aminoacids are alanine (A), cysteine (C), aspartic acid (D), glycine (G),asparagine (N), proline (P), serine (S), threonine (T), and valine (V).Very small amino acids are alanine (A), glycine (G) and serine (S).Amino acids likely to be involved in a turn formation are alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), glycine (G),histidine (H), lysine (K), asparagine (N), glutamine (Q), arginine (R),serine (S), proline (P), and threonine (T). As non-limiting examples,the amino acids within each of these defined groups may be substitutedfor each other in the formulas described above, as conservativesubstitutions, subject to the specific preferences stated herein.

Substantial changes in function can be made by selecting substitutionsthat are less conservative than those shown in the defined groups,above. For example, non-conservative substitutions can be made whichmore significantly affect the structure of the peptide in the area ofthe alteration, for example, the alpha-helical, or beta-sheet structure;the charge or hydrophobicity of the molecule at the target site; or thebulk of the side chain. The substitutions which generally are expectedto produce the greatest changes in the peptide's properties are thosewhere 1) a hydrophilic residue, e.g., seryl or threonyl, is substitutedfor (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl, or alanyl; 2) a cysteine or proline is substitutedfor (or by) any other residue; 3) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or 4) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) a residue that does not have a side chain, e.g., glycine.

Amino acid preferences have been identified for certain peptides andpeptide groups of the present invention. For example, amino acidpreferences for the RP9, D8, and Group 6 (Formula 10) peptides are shownin Tables 17-19, below. In some instances, cysteine pairs may also bepreferred. For example, cysteine pairs are preferred in certain Formula1 and Formula 2 sequences described herein. In accordance with theinvention, the amino acid sequences of the invention may include two ormore cysteine residues, which may be separated by at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids, and maybe positioned inside or outside the Formula 1 or Formula 2 motifsequence. Preferably, the cysteines are separated by 17 or 18 aminoacids.

Variants also include amino acid sequences in which one or more residuesare modified (i.e., by phosphorylation, sulfation, acylation,PEGylation, etc.), and mutants comprising one or more modified residues.Amino acid sequences may also be modified with a label capable ofproviding a detectable signal, either directly or indirectly, including,but not limited to, radioisotope, fluorescent, and enzyme labels.Fluorescent labels include, for example, Cy₃, Cy₅, Alexa, BODIPY,fluorescein (e.g., FluorX, DTAF, and FITC), rhodamine (e.g., TRITC),auramine, Texas Red, AMCA blue, and Lucifer Yellow. Preferred isotopelabels include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y,¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Preferred enzyme labels include peroxidase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plus peroxidase, and alkaline phosphatase (see, e.g., U.S. Pat.Nos. 3,654,090; 3,850,752 and 4,016,043). Enzymes can be conjugated byreaction with bridging molecules such as carbodiimides, diisocyanates,glutaraldehyde, and the like. Enzyme labels can be detected visually, ormeasured by calorimetric, spectrophotometric, fluorospectrophotometric,amperometric, or gasometric techniques. Other labeling systems, such asavidin/biotin, Tyramide Signal Amplification (TSA™), are known in theart, and are commercially available (see, e.g., ABC kit, VectorLaboratories, Inc., Burlingame, Calif.; NEN® Life Science Products,Inc., Boston, Mass.).

1. Recombinant Synthesis of Peptides

To obtain recombinant peptides, DNA sequences encoding these peptidesmay be cloned into any suitable vectors for expression in intact hostcells or in cell-free translation systems by methods well-known in theart (see Sambrook et al., 1989). The particular choice of the vector,host, or translation system is not critical to the practice of theinvention.

A large number of vectors, including bacterial, yeast, and mammalianvectors, have been described for replication and/or expression invarious host cells or cell-free systems, and may be used for genetherapy as well as for simple cloning or protein expression. In oneaspect of the present invention, an expression vector comprises anucleic acid encoding a IR or IGF-1R agonist or antagonist peptide, asdescribed herein, operably linked to at least one regulatory sequence.Regulatory sequences are known in the art and are selected to directexpression of the desired protein in an appropriate host cell.Accordingly, the term regulatory sequence includes promoters, enhancersand other expression control elements (see D. V. Goeddel (1990) MethodsEnzymol. 185:3-7). Enhancer and other expression control sequences aredescribed in Enhancers and Eukaryotic Gene Expression, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1983). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell to be transfected and/or the type of peptidedesired to be expressed.

Several regulatory elements (e.g., promoters) have been isolated andshown to be effective in the transcription and translation ofheterologous proteins in the various hosts. Such regulatory regions,methods of isolation, manner of manipulation, etc. are known in the art.Non-limiting examples of bacterial promoters include the β-lactamase(penicillinase) promoter; lactose promoter; tryptophan (trp) promoter;araBAD (arabinose) operon promoter; lambda-derived P₁ promoter and Ngene ribosome binding site; and the hybrid tac promoter derived fromsequences of the trp and lac UV5 promoters. Non-limiting examples ofyeast promoters include the 3-phosphoglycerate kinase promoter,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, galactokinase(GAL1) promoter, galactoepimerase promoter, and alcohol dehydrogenase(ADH1) promoter. Suitable promoters for mammalian cells include, withoutlimitation, viral promoters, such as those from Simian Virus 40 (SV40),Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus(BPV). Preferred replication and inheritance systems include M13, ColE1,SV40, baculovirus, lambda, adenovirus, CEN ARS, 2 μm ARS and the like.While expression vectors may replicate autonomously, they may alsoreplicate by being inserted into the genome of the host cell, by methodswell-known in the art.

To obtain expression in eukaryotic cells, terminator sequences,polyadenylation sequences, and enhancer sequences that modulate geneexpression may be required. Sequences that cause amplification of thegene may also be desirable. Furthermore, sequences that facilitatesecretion of the recombinant product from cells, including, but notlimited to, bacteria, yeast, and animal cells, such as secretory signalsequences and/or preprotein or proprotein sequences, may also beincluded. These sequences are well-described in the art. DNA sequencescan be optimized, if desired, for more efficient expression in a givenhost organism or expression system. For example, codons can be alteredto conform to the preferred codon usage in a given host cell orcell-free translation system using well-established techniques.

Codon usage data can be obtained from publicly-available sources, forexample, the Codon Usage Database at http://www.kazusa.or.jp/codon/. Inaddition, computer programs that translate amino acid sequenceinformation into nucleotide sequence information in accordance withcodon preferences (i.e., backtranslation programs) are widely available.See, for example, Backtranslate program from Genetics Computer Group(GCG), Accelrys, Inc., Madison, Wis.; and Backtranslation Applet fromEntelechon GmbH, Regensburg, Germany. Thus, using the peptide sequencesdisclosed herein, one of ordinary skill in the art can design nucleicacids to yield optimal expression levels in the translation system orhost cell of choice.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells that express the inserts. Typicalselection genes encode proteins that 1) confer resistance to antibioticsor other toxic substances, e.g., ampicillin, neomycin, methotrexate,etc.; 2) complement auxotrophic deficiencies, or 3) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. Markers may be an inducible ornon-inducible gene and will generally allow for positive selection.Non-limiting examples of markers include the ampicillin resistancemarker (i.e., beta-lactamase), tetracycline resistance marker,neomycin/kanamycin resistance marker (i.e., neomycinphosphotransferase), dihydrofolate reductase, glutamine synthetase, andthe like. The choice of the proper selectable marker will depend on thehost cell, and appropriate markers for different hosts as understood bythose of skill in the art.

Suitable expression vectors for use with the present invention include,but are not limited to, pUC, pBluescript (Stratagene), pET (Novagen,Inc., Madison, Wis.), and pREP (Invitrogen) plasmids. Vectors cancontain one or more replication and inheritance systems for cloning orexpression, one or more markers for selection in the host, e.g.,antibiotic resistance, and one or more expression cassettes. Theinserted coding sequences can be synthesized by standard methods,isolated from natural sources, or prepared as hybrids. Ligation of thecoding sequences to transcriptional regulatory elements (e.g.,promoters, enhancers, and/or insulators) and/or to other amino acidencoding sequences can be carried out using established methods.

Suitable cell-free expression systems for use with the present inventioninclude, without limitation, rabbit reticulocyte lysate, wheat germextract, canine pancreatic microsomal membranes, E. Coli S30 extract,and coupled transcription/translation systems (Promega Corp., Madison,Wis.). These systems allow the expression of recombinant peptides uponthe addition of cloning vectors, DNA fragments, or RNA sequencescontaining protein-coding regions and appropriate promoter elements.

Non-limiting examples of suitable host cells include bacteria, archea,insect, fungi (e.g., yeast), plant, and animal cells (e.g., mammalian,especially human). Of particular interest are Escherichia coli, Bacillussubtilis, Saccharomyces cerevisiae, SF9 cells, C129 cells, 293 cells,Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.Techniques for the propagation of mammalian cells in culture arewell-known (see, Jakoby and Pastan (Eds), 1979, Cell Culture. Methods inEnzymology, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich,N.Y.). Examples of commonly used mammalian host cell lines are VERO andHeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although itwill be appreciated by the skilled practitioner that other cell linesmay be used, e.g., to provide higher expression, or other features.

Host cells can be transformed, transfected, or infected as appropriateby any suitable method including electroporation, calcium chloride-,lithium chloride-, lithium acetate/polyethylene glycol-, calciumphosphate-, DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting,injection, microinjection, microprojectile bombardment, phage infection,viral infection, or other established methods. Alternatively, vectorscontaining the nucleic acids of interest can be transcribed in vitro,and the resulting RNA introduced into the host cell by well-knownmethods, e.g., by injection (see, Kubo et al., 1988, FEBS Letts.241:119). The cells into which have been introduced nucleic acidsdescribed above are meant to also include the progeny of such cells.

Nucleic acids encoding the peptides of the invention may be isolateddirectly from recombinant phage libraries (e.g., RAPIDLIB® or GRABLIB®libraries) described herein. Alternatively, the polymerase chainreaction (PCR) method can be used to produce nucleic acids of theinvention, using the recombinant phage libraries as templates. Primersused for PCR can be synthesized using the sequence information providedherein and can further be designed to introduce appropriate newrestriction sites, if desirable, to facilitate incorporation into agiven vector for recombinant expression.

Nucleic acids encoding the peptides of the present invention can also beproduced by chemical synthesis, e.g., by the phosphoramidite methoddescribed by Beaucage et al., 1981, Tetra. Letts. 22:1859-1862, or thetriester method according to Matteucci et al., 1981, J. Am. Chem. Soc.,103:3185, and can performed on commercial, automated oligonucleotidesynthesizers. A double-stranded fragment may be obtained from thesingle-stranded product of chemical synthesis either by synthesizing thecomplementary strand and annealing the strands together underappropriate conditions or by adding the complementary strand using DNApolymerase with an appropriate primer sequence.

The nucleic acids encoding the peptides of the invention can be producedin large quantities by replication in a suitable host cell. Natural orsynthetic nucleic acid fragments, comprising at least ten contiguousbases coding for a desired amino acid sequence can be incorporated intorecombinant nucleic acid constructs, usually DNA constructs, capable ofintroduction into and replication in a prokaryotic or eukaryotic cell.Usually the nucleic acid constructs will be suitable for replication ina unicellular host, such as yeast or bacteria, but may also be intendedfor introduction to (with and without integration within the genome)cultured mammalian or plant or other eukaryotic cells, cell lines,tissues, or organisms. The purification of nucleic acids produced by themethods of the present invention is described, for example, in Sambrooket al., 1989; F. M. Ausubel et al., 1992, Current Protocols in MolecularBiology, J. Wiley and Sons, New York, N.Y.

These nucleic acids can encode variant or truncated forms of thepeptides as well as the reference peptides shown in FIGS. 1-4, 8, and 9and Table 7, inter alia. Large quantities of the nucleic acids andpeptides of the present invention may be prepared by expressing thenucleic acids or portions thereof in vectors or other expressionvehicles in compatible prokaryotic or eukaryotic host cells. The mostcommonly used prokaryotic hosts are strains of Escherichia coli,although other prokaryotes, such as Bacillus subtilis or Pseudomonas mayalso be used. Mammalian or other eukaryotic host cells, such as those ofyeast, filamentous fungi, plant, insect, or amphibian or avian species,may also be useful for production of the proteins of the presentinvention. For example, insect cell systems (i.e., lepidopteran hostcells and baculovirus expression vectors) are particularly suited forlarge-scale protein production.

Host cells carrying an expression vector (i.e., transformants or clones)are selected using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule,preferably the same DNA molecule. In prokaryotic hosts, the transformantmay be selected, e.g., by resistance to ampicillin, tetracycline orother antibiotics. Production of a particular product based ontemperature sensitivity may also serve as an appropriate marker.

For some purposes, it is preferable to produce the peptide in arecombinant system in which the peptide contains an additional sequence(e.g., epitope or protein) tag that facilitates purification.Non-limiting examples of epitope tags include c-myc, haemagglutinin(HA), polyhistidine (6X-HIS) (SEQ ID NO:1778), GLU-GLU, and DYKDDDDK(SEQ ID NO:1779) or DYKD (SEQ ID NO:1545; FLAG®) epitope tags.Non-limiting examples of protein tags include glutathione-S-transferase(GST), green fluorescent protein (GFP), and maltose binding protein(MBP). In one approach, the coding sequence of a peptide can be clonedinto a vector that creates a fusion with a sequence tag of interest.Suitable vectors include, without limitation, PRSET (Invitrogen Corp.,San Diego, Calif.), pGEX (Amersham Pharmacia Biotech, Inc., Piscataway,N.J.), pEGFP (CLONTECH Laboratories, Inc., Palo Alto, Calif.), and pMAL™(New England BioLabs, Inc., Beverly, Mass.) plasmids. Followingexpression, the epitope or protein tagged peptide can be purified from acrude lysate of the translation system or host cell by chromatography onan appropriate solid-phase matrix. In some cases, it may be preferableto remove the epitope or protein tag (i.e., via protease cleavage)following purification.

Methods for directly purifying peptides from sources such as cellular orextracellular lysates are well-known in the art (see Harris and Angal,1989). Such methods include, without limitation, sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE),preparative disc-gel electrophoresis, isoelectric focusing,high-performance liquid chromatography (HPLC), reversed-phase HPLC, gelfiltration, ion exchange and partition chromatography, countercurrentdistribution, and combinations thereof. Peptides can be purified frommany possible sources, for example, plasma, body tissues, or body fluidlysates derived from human or animal, including mammalian, bird, fish,and insect sources.

Antibody-based methods may also be used to purify peptides. Antibodiesthat recognize these peptides or fragments derived therefrom can beproduced and isolated. The peptide can then be purified from a crudelysate by chromatography on an antibody-conjugated solid-phase matrix(see Harlow and Lane, 1998).

2. Chemical Synthesis of Peptides

Alternately, peptides may be chemically synthesized by commerciallyavailable automated procedures, including, without limitation, exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. The peptides arepreferably prepared by solid-phase peptide synthesis; for example, asdescribed by Merrifield (1965; 1997).

According to methods known in the art, peptides can be chemicallysynthesized by commercially available automated procedures, including,without limitation, exclusive solid phase synthesis, partial solid phasemethods, fragment condensation, classical solution synthesis. Inaddition, recombinant and synthetic methods of peptide production can becombined to produce semi-synthetic peptides. The peptides of theinvention are preferably prepared by solid phase peptide synthesis asdescribed by Merrifield, 1963, J. Am. Chem. Soc. 85:2149; 1997. In oneembodiment, synthesis is carried out with amino acids that are protectedat the alpha-amino terminus. Trifunctional amino acids with labileside-chains are also protected with suitable groups to prevent undesiredchemical reactions from occurring during the assembly of the peptides.The alpha-amino protecting group is selectively removed to allowsubsequent reaction to take place at the amino-terminus. The conditionsfor the removal of the alpha-amino protecting group do not remove theside-chain protecting groups.

The alpha-amino protecting groups are those known to be useful in theart of stepwise peptide synthesis. Included are acyl type protectinggroups, e.g., formyl, trifluoroacetyl, acetyl, aromatic urethane typeprotecting groups, e.g., benzyloxycarbonyl (Cbz), substitutedbenzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl (Fmoc), aliphaticurethane protecting groups, e.g., t-butyloxycarbonyl (Boc),isopropyloxycarbonyl, cyclohexyloxycarbonyl, and alkyl type protectinggroups, e.g., benzyl, triphenylmethyl. The preferred protecting group isBoc. The side-chain protecting groups for Tyr include tetrahydropyranyl,tert-butyl, trityl, benzyl, Cbz, 4-Br-Cbz and 2,6-dichlorobenzyl. Thepreferred side-chain protecting group for Tyr is 2,6-dichlorobenzyl. Theside-chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl,methyl, ethyl, and cyclohexyl. The preferred side-chain protecting groupfor Asp is cyclohexyl. The side-chain protecting groups for Thr and Serinclude acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl,2,6-dichlorobenzyl, and Cbz. The preferred protecting group for Thr andSer is benzyl. The side-chain protecting groups for Arg include nitro,Tos, Cbz, adamantyloxycarbonyl, and Boc. The preferred protecting groupfor Arg is Tos. The side-chain amino group of Lys can be protected withCbz, 2-Cl-Cbz, Tos, or Boc. The 2-Cl-Cbz group is the preferredprotecting group for Lys.

The side-chain protecting groups selected must remain intact duringcoupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis, using reaction conditions that will not alter the finishedpeptide.

Solid phase synthesis is usually carried out from the carboxy-terminusby coupling the alpha-amino protected (side-chain protected) amino acidto a suitable solid support. An ester linkage is formed when theattachment is made to a chloromethyl or hydroxymethyl resin, and theresulting peptide will have a free carboxyl group at the C-terminus.Alternatively, when a benzhydrylamine or p-methylbenzhydrylamine resinis used, an amide bond is formed and the resulting peptide will have acarboxamide group at the C-terminus. These resins are commerciallyavailable, and their preparation has described by Stewart et al., 1984,Solid Phase Peptide Synthesis (2nd Edition), Pierce Chemical Co.,Rockford, Ill.

The C-terminal amino acid, protected at the side chain if necessary andat the alpha-amino group, is coupled to the benzhydrylamine resin usingvarious activating agents including dicyclohexylcarbodiimide (DCC),N,N′-diisopropyl-carbodiimide and carbonyldiimidazole. Following theattachment to the resin support, the alpha-amino protecting group isremoved using trifluoroacetic acid (TFA) or HCl in dioxane at atemperature between 0 and 25° C. Dimethylsulfide is added to the TFAafter the introduction of methionine (Met) to suppress possibleS-alkylation. After removal of the alpha-amino protecting group, theremaining protected amino acids are coupled stepwise in the requiredorder to obtain the desired sequence.

Various activating agents can be used for the coupling reactionsincluding DCC,N,N′-diisopropyl-carbodiimide,benzotriazol-1-yl-oxy-tris-(dimethylamino) phosphoniumhexa-fluorophosphate (BOP) and DCC-hydroxybenzotriazole (HOBt). Eachprotected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH₂Cl₂ or mixtures thereof. The extent of completion of thecoupling reaction is monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., 1970, Anal. Biochem. 34:595. Incases where incomplete coupling is found, the coupling reaction isrepeated. The coupling reactions can be performed automatically withcommercially available instruments.

After the entire assembly of the desired peptide, the peptide-resin iscleaved with a reagent such as liquid HF for 1-2 h at 0° C., whichcleaves the peptide from the resin and removes all side-chain protectinggroups. A scavenger such as anisole is usually used with the liquid HFto prevent cations formed during the cleavage from alkylating the aminoacid residues present in the peptide. The peptide-resin can bedeprotected with TFA/dithioethane prior to cleavage if desired.

Side-chain to side-chain cyclization on the solid support requires theuse of an orthogonal protection scheme which enables selective cleavageof the side-chain functions of acidic amino acids (e.g., Asp) and thebasic amino acids (e.g., Lys). The 9-fluorenylmethyl (Fm) protectinggroup for the side-chain of Asp and the 9-fluorenylmethyloxycarbonyl(Fmoc) protecting group for the side-chain of Lys can be used for thispurpose. In these cases, the side-chain protecting groups of theBoc-protected peptide-resin are selectively removed with piperidine inDMF. Cyclization is achieved on the solid support using variousactivating agents including DCC, DCC/HOBt, or BOP. The HF reaction iscarried out on the cyclized peptide-resin as described above.

3. Peptide Libraries

Peptide libraries produced and screened according to the presentinvention are useful in providing new ligands for IR and IGF-1R. Peptidelibraries can be designed and panned according to methods described indetail herein, and methods generally available to those in the art (see,e.g., U.S. Pat. No. 5,723,286 issued Mar. 3, 1998 to Dower et al.). Inone aspect, commercially available phage display libraries can be used(e.g., RAPIDLIB® or GRABLIB®, DGI BioTechnologies, Inc., Edison, N.J.;Ph.D. C7C Disulfide Constrained Peptide Library, New England Biolabs).In another aspect, an oligonucleotide library can be prepared accordingto methods known in the art, and inserted into an appropriate vector forpeptide expression. For example, vectors encoding a bacteriophagestructural protein, preferably an accessible phage protein, such as abacteriophage coat protein, can be used. Although one skilled in the artwill appreciate that a variety of bacteriophage may be employed in thepresent invention, in preferred embodiments the vector is, or is derivedfrom, a filamentous bacteriophage, such as, for example, f1, fd, Pf1,M13, etc. In particular, the fd-tet vector has been extensivelydescribed in the literature (see, e.g., Zacher et al., 1980, Gene9:127-140; Smith et al., 1985, Science 228:1315-1317; Parmley and Smith,1988, Gene 73:305-318).

The phage vector is chosen to contain or is constructed to contain acloning site located in the 5′ region of the gene encoding thebacteriophage structural protein, so that the peptide is accessible toreceptors in an affinity enrichment procedure as described hereinbelow.The structural phage protein is preferably a coat protein. An example ofan appropriate coat protein is pIII. A suitable vector may alloworiented cloning of the oligonucleotide sequences that encode thepeptide so that the peptide is expressed at or within a distance ofabout 100 amino acid residues of the N-terminus of the mature coatprotein. The coat protein is typically expressed as a preprotein, havinga leader sequence.

Thus, desirably the oligonucleotide library is inserted so that theN-terminus of the processed bacteriophage outer protein is the firstresidue of the peptide, i.e., between the 3′-terminus of the sequenceencoding the leader protein and the 5′-terminus of the sequence encodingthe mature protein or a portion of the 5′ terminus. The library isconstructed by cloning an oligonucleotide which contains the variableregion of library members (and any spacers, as discussed below) into theselected cloning site. Using known recombinant DNA techniques (seegenerally, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), an oligonucleotide may be constructed which, inter alia; 1)removes unwanted restriction sites and adds desired ones; 2)reconstructs the correct portions of any sequences which have beenremoved (such as a correct signal peptidase site, for example); 3)inserts the spacer residues, if any; and/or 4) corrects the translationframe (if necessary) to produce active, infective phage.

The central portion of the oligonucleotide will generally contain one ormore IR and/or IGF-1R binding sequences and, optionally, spacersequences. The sequences are ultimately expressed as peptides (with orwithout spacers) fused to or in the N-terminus of the mature coatprotein on the outer, accessible surface of the assembled bacteriophageparticles. The size of the library will vary according to the number ofvariable codons, and hence the size of the peptides, which are desired.Generally the library will be at least about 10⁶ members, usually atleast 10⁷, and typically 10⁸ or more members. To generate the collectionof oligonucleotides which forms a series of codons encoding a randomcollection of amino acids and which is ultimately cloned into thevector, a codon motif is used, such as (NNK)_(x), where N may be A, C,G, or T (nominally equimolar), K is G or T (nominally equimolar), and xis typically up to about 5, 6, 7, 8, or more, thereby producinglibraries of penta-, hexa-, hepta-, and octa-peptides or larger. Thethird position may also be G or C, designated “S”. Thus, NNK or NNS 1)code for all the amino acids; 2) code for only one stop codon; and 3)reduce the range of codon bias from 6:1 to 3:1.

It should be understood that, with longer peptides, the size of thelibrary that is generated may become a constraint in the cloningprocess. The expression of peptides from randomly generated mixtures ofoligonucleotides in appropriate recombinant vectors is known in the art(see, e.g., Oliphant et al., Gene 44:177-183). For example, the codonmotif (NNK)₆ produces 32 codons, one for each of 12 amino acids, two foreach of five amino acids, three for each of three amino acids and one(amber) stop codon. Although this motif produces a codon distribution asequitable as available with standard methods of oligonucleotidesynthesis, it results in a bias against peptides containing one-codonresidues. In particular, a complete collection of hexacodons containsone sequence encoding each peptide made up of only one-codon aminoacids, but contains 729 (3⁶) sequences encoding each peptide with onlythree-codon amino acids.

An alternative approach to minimize the bias against one-codon residuesinvolves the synthesis of 20 activated trinucleotides, each representingthe codon for one of the 20 genetically encoded amino acids. These aresynthesized by conventional means, removed from the support whilemaintaining the base and 5-OH-protecting groups, and activated by theaddition of 3′O-phosphoramidite (and phosphate protection withb-cyanoethyl groups) by the method used for the activation ofmononucleosides (see, generally, McBride and Caruthers, 1983,Tetrahedron Letters 22:245). Degenerate oligocodons are prepared usingthese trimers as building blocks. The trimers are mixed at the desiredmolar ratios and installed in the synthesizer. The ratios will usuallybe approximately equimolar, but may be a controlled unequal ratio toobtain the over- to under-representation of certain amino acids codedfor by the degenerate oligonucleotide collection. The condensation ofthe trimers to form the oligocodons is done essentially as described forconventional synthesis employing activated mononucleosides as buildingblocks (see, e.g., Atkinson and Smith, 1984, Oligonucleotide Synthesis,M. J. Gait, Ed., p. 35-82). This procedure generates a population ofoligonucleotides for cloning that is capable of encoding an equaldistribution (or a controlled unequal distribution) of the possiblepeptide sequences. Advantageously, this approach may be employed ingenerating longer peptide sequences, since the range of bias produced bythe (NNK)₆ motif increases by three-fold with each additional amino acidresidue.

When the codon motif is (NNK)_(x), as defined above, and when x equals8, there are 2.6×10¹⁰ possible octa-peptides. A library containing mostof the octa-peptides may be difficult to produce. Thus, a sampling ofthe octa-peptides may be accomplished by constructing a subset libraryusing up to about 10% of the possible sequences, which subset ofrecombinant bacteriophage particles is then screened. If desired, toextend the diversity of a subset library, the recovered phage subset maybe subjected to mutagenesis and then subjected to subsequent rounds ofscreening. This mutagenesis step may be accomplished in two generalways: the variable region of the recovered phage may be mutagenized, oradditional variable amino acids may be added to the regions adjoiningthe initial variable sequences.

To diversify around active peptides (i.e., binders) found in earlyrounds of panning, the positive phage can sequenced to determine theidentity of the active peptides. Oligonucleotides can then besynthesized based on these peptide sequences. The syntheses are donewith a low level of all bases incorporated at each step to produceslight variations of the primary oligonucleotide sequences. This mixtureof (slightly) degenerate oligonucleotides can then be cloned into theaffinity phage by methods known to those in the art. This methodproduces systematic, controlled variations of the starting peptidesequences as part of a secondary library. It requires, however, thatindividual positive phage be sequenced before mutagenesis, and thus isuseful for expanding the diversity of small numbers of recovered phage.

An alternate approach to diversify the selected phage allows themutagenesis of a pool, or subset, of recovered phage. In accordance withthis approach, phage recovered from panning are pooled and singlestranded DNA is isolated. The DNA is mutagenized by treatment with,e.g., nitrous acid, formic acid, or hydrazine. These treatments producea variety of damage to the DNA. The damaged DNA is then copied withreverse transcriptase, which misincorporates bases when it encounters asite of damage. The segment containing the sequence encoding thereceptor-binding peptide is then isolated by cutting with restrictionnuclease(s) specific for sites flanking the peptide coding sequence.This mutagenized segment is then recloned into undamaged vector DNA, theDNA is transformed into cells, and a secondary library according toknown methods. General mutagenesis methods are known in the art (seeMyers et al., 1985, Nucl. Acids Res. 13:3131-3145; Myers et al., 1985,Science 229:242-246; Myers, 1989, Current Protocols in Molecular BiologyVol. I, 8.3.1-8.3.6, F. Ausubel et al., eds, J. Wiley and Sons, NewYork).

In another general approach, the addition of amino acids to a peptide orpeptides found to be active, can be carried out using various methods.In one, the sequences of peptides selected in early panning aredetermined individually and new oligonucleotides, incorporating thedetermined sequence and an adjoining degenerate sequence, aresynthesized. These are then cloned to produce a secondary library.Alternatively, methods can be used to add a second IR or IGF-1R bindingsequence to a pool of peptide-bearing phage. In accordance with onemethod, a restriction site is installed next to the first IR or IGF-1Rbinding sequence. Preferably, the enzyme should cut outside of itsrecognition sequence. The recognition site may be placed several basesfrom the first binding sequence. To insert a second IR or IGF-1R bindingsequence, the pool of phage DNA is digested and blunt-ended by fillingin the overhang with Klenow fragment. Double-stranded, blunt-ended,degenerately synthesized oligonucleotides are then ligated into thissite to produce a second binding sequence juxtaposed to the firstbinding sequence. This secondary library is then amplified and screenedas before.

While in some instances it may be appropriate to synthesize longerpeptides to bind certain receptors, in other cases it may be desirableto provide peptides having two or more IR or IGF-1R binding sequencesseparated by spacer (e.g., linker) residues. For example, the bindingsequences may be separated by spacers that allow the regions of thepeptides to be presented to the receptor in different ways. The distancebetween binding regions may be as little as 1 residue, or at least 2-20residues, or up to at least 100 residues. Preferred spacers are 3, 6, 9,12, 15, or 18 residues in length. For probing large binding sites ortandem binding sites (e.g., Site 1 and Site 2 of IR), the bindingregions may be separated by a spacer of residues of up to 20 to 30 aminoacids. The number of spacer residues when present will typically be atleast 2 residues, and often will be less than 20 residues.

The oligonucleotide library may have binding sequences which areseparated by spacers (e.g., linkers), and thus may be represented by theformula: (NNK)_(y)−(abc)_(n)−(NNK)_(z) where N and K are as definedpreviously (note that S as defined previously may be substituted for K),and y+z is equal to about 5, 6, 7, 8, or more, a, b and c represent thesame or different nucleotides comprising a codon encoding spacer aminoacids, n is up to about 3, 6, 9, or 12 amino acids, or more. The spacerresidues may be somewhat flexible, comprising oligo-glycine, oroligo-glycine-glycine-serine, for example, to provide the diversitydomains of the library with the ability to interact with sites in alarge binding site relatively unconstrained by attachment to the phageprotein. Rigid spacers, such as, e.g., oligo-proline, may also beinserted separately or in combination with other spacers, includingglycine spacers. It may be desired to have the IR or IGF-1R bindingsequences close to one another and use a spacer to orient the bindingsequences with respect to each other, such as by employing a turnbetween the two sequences, as might be provided by a spacer of thesequence glycine-proline-glycine, for example. To add stability to sucha turn, it may be desirable or necessary to add cysteine residues ateither or both ends of each variable region. The cysteine residues wouldthen form disulfide bridges to hold the variable regions together in aloop, and in this fashion may also serve to mimic a cyclic peptide. Ofcourse, those skilled in the art will appreciate that various othertypes of covalent linkages for cyclization may also be used.

Spacer residues as described above may also be situated on either orboth ends of the IR or IGF-1R binding sequences. For instance, a cyclicpeptide may be designed without an intervening spacer, by having acysteine residue on both ends of the peptide. As described above,flexible spacers, e.g., oligo-glycine, may facilitate interaction of thepeptide with the selected receptors. Alternatively, rigid spacers mayallow the peptide to be presented as if on the end of a rigid arm, wherethe number of residues, e.g., proline residues, determines not only thelength of the arm but also the direction for the arm in which thepeptide is oriented. Hydrophilic spacers, made up of charged and/oruncharged hydrophilic amino acids, (e.g., Thr, H is, Asn, Gln, Arg, Glu,Asp, Met, Lys, etc.), or hydrophobic spacers of hydrophobic amino acids(e.g., Phe, Leu, Ile, Gly, Val, Ala, etc.) may be used to present thepeptides to receptor binding sites with a variety of local environments.

Notably, some peptides, because of their size and/or sequence, may causesevere defects in the infectivity of their carrier phage. This causes aloss of phage from the population during reinfection and amplificationfollowing each cycle of panning. To minimize problems associated withdefective infectivity, DNA prepared from the eluted phage can betransformed into appropriate host cells, such as, e.g., E. coli,preferably by electroporation (see, e.g., Dower et al., Nucl. Acids Res.16:6127-6145), or well-known chemical means. The cells are cultivatedfor a period of time sufficient for marker expression, and selection isapplied as typically done for DNA transformation. The colonies areamplified, and phage harvested for affinity enrichment in accordancewith established methods. Phage identified in the affinity enrichmentmay be re-amplified by infection into the host cells. The successfultransformants are selected by growth in an appropriate antibiotic(s),e.g., tetracycline or ampicillin. This may be done on solid or in liquidgrowth medium.

For growth on solid medium, the cells are grown at a high density (about10⁸ to 10⁹ transformants per m²) on a large surface of, for example,L-agar containing the selective antibiotic to form essentially aconfluent lawn. The cells and extruded phage are scraped from thesurface and phage are prepared for the first round of panning (see,e.g., Parmley and Smith, 1988, Gene 73:305-318). For growth in liquidculture, cells may be grown in L-broth and antibiotic through about 10or more doublings. The phage are harvested by standard procedures (seeSambrook et al., 1989, Molecular Cloning, 2^(nd) ed.). Growth in liquidculture may be more convenient because of the size of the libraries,while growth on solid media likely provides less chance of bias duringthe amplification process.

For affinity enrichment of desired clones, generally about 10³ to 10⁴library equivalents (a library equivalent is one of each recombinant;10⁴ equivalents of a library of 10⁹ members is 10⁹×10⁴=10¹³ phage), buttypically at least 102 library equivalents, up to about 10⁵ to 10⁶, areincubated with a receptor (or portion thereof) to which the desiredpeptide is sought. The receptor is in one of several forms appropriatefor affinity enrichment schemes. In one example the receptor isimmobilized on a surface or particle, and the library of phage bearingpeptides is then panned on the immobilized receptor generally accordingto procedures known in the art. In an alternate scheme, a receptor isattached to a recognizable ligand (which may be attached via a tether).A specific example of such a ligand is biotin. The receptor, somodified, is incubated with the library of phage and binding occurs withboth reactants in solution. The resulting complexes are then bound tostreptavidin (or avidin) through the biotin moiety. The streptavidin maybe immobilized on a surface such as a plastic plate or on particles, inwhich case the complexes (phage/peptide/receptor/biotin/streptavidin)are physically retained; or the streptavidin may be labeled, with afluorophor, for example, to tag the active phage/peptide for detectionand/or isolation by sorting procedures, e.g., on afluorescence-activated cell sorter.

Phage that associate with IR or IGF-1R via non-specific interactions areremoved by washing. The degree and stringency of washing required willbe determined for each receptor/peptide of interest. A certain degree ofcontrol can be exerted over the binding characteristics of the peptidesrecovered by adjusting the conditions of the binding incubation and thesubsequent washing. The temperature, pH, ionic strength, divalent cationconcentration, and the volume and duration of the washing will selectfor peptides within particular ranges of affinity for the receptor.Selection based on slow dissociation rate, which is usually predictiveof high affinity, is the most practical route. This may be done eitherby continued incubation in the presence of a saturating amount of freeligand, or by increasing the volume, number, and length of the washes.In each case, the rebinding of dissociated peptide-phage is prevented,and with increasing time, peptide-phage of higher and higher affinityare recovered. Additional modifications of the binding and washingprocedures may be applied to find peptides that bind receptors underspecial conditions. Once a peptide sequence that imparts some affinityand specificity for the receptor molecule is known, the diversity aroundthis binding motif may be embellished. For instance, variable peptideregions may be placed on one or both ends of the identified sequence.The known sequence may be identified from the literature, or may bederived from early rounds of panning in the context of the presentinvention.

G. Screening Assays

In another embodiment of this invention, screening assays to identifypharmacologically active ligands at IR and/or IGF-1R are provided.Ligands may encompass numerous chemical classes, though typically theyare organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons. Suchligands can comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. Ligands oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Ligands can also comprise biomolecules includingpeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs, or combinations thereof.

Ligands may include, for example, 1) peptides such as soluble peptides,including Ig-tailed fusion peptides and members of random peptidelibraries (see, e.g., Lam et al., 1991, Nature 354:82-84; Houghten etal., 1991, Nature 354:84-86) and combinatorial chemistry-derivedmolecular libraries made of D- and/or L-configuration amino acids; 2)phosphopeptides (e.g., members of random and partially degenerate,directed phosphopeptide libraries, see, e.g., Songyang et al, 1993, Cell72:767-778); 3) antibodies (e.g., polyclonal, monoclonal, humanized,anti-idiotypic, chimeric, and single chain antibodies as well as Fab,F(ab′)₂, Fab expression library fragments, and epitope-binding fragmentsof antibodies); and 4) small organic and inorganic molecules.

Ligands can be obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. Synthetic compoundlibraries are commercially available from, for example, MaybridgeChemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.),Brandon Associates (Merrimack, N.H.), and Microsource (New Milford,Conn.). A rare chemical library is available from Aldrich ChemicalCompany, Inc. (Milwaukee, Wis.). Natural compound libraries comprisingbacterial, fungal, plant or animal extracts are available from, forexample, Pan Laboratories (Bothell, Wash.). In addition, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides.

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts can be readily produced. Methods forthe synthesis of molecular libraries are readily available (see, e.g.,DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al.,1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al., 1994, J.Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carell et al.,1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew.Chem. Int. Ed. Engl. 33:2061; and in Gallop et al., 1994, J. Med. Chem.37:1233). In addition, natural or synthetic compound libraries andcompounds can be readily modified through conventional chemical,physical and biochemical means (see, e.g., Blondelle et al., 1996,Trends in Biotech. 14:60), and may be used to produce combinatoriallibraries. In another approach, previously identified pharmacologicalagents can be subjected to directed or random chemical modifications,such as acylation, alkylation, esterification, amidification, and theanalogs can be screened for IR-modulating activity.

Numerous methods for producing combinatorial libraries are known in theart, including those involving biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide or peptide libraries, while the other four approaches areapplicable to polypeptide, peptide, non-peptide oligomer, or smallmolecule libraries of compounds (K. S. Lam, 1997, Anticancer Drug Des.12:145).

Libraries may be screened in solution by methods generally known in theart for determining whether ligands competitively bind at a commonbinding site. Such methods may including screening libraries in solution(e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam,1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),bacteria or spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull etal., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869), or on phage (Scottand Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406;Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 97:6378-6382; Felici,1991, J. Mol. Biol. 222:301-310; Ladner, supra).

Where the screening assay is a binding assay, IR, or one of theIR-binding peptides disclosed herein, may be joined to a label, wherethe label can directly or indirectly provide a detectable signal.Various labels include radioisotopes, fluorescent molecules,chemiluminescent molecules, enzymes, specific binding molecules,particles, e.g., magnetic particles, and the like. Specific bindingmolecules include pairs, such as biotin and streptavidin, digoxin andantidigoxin, etc. For the specific binding members, the complementarymember would normally be labeled with a molecule that provides fordetection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc., which are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc., may be used. Thecomponents are added in any order that produces the requisite binding.Incubations are performed at any temperature that facilitates optimalactivity, typically between 4° and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Normally, between 0.1 and 1 h will besufficient. In general, a plurality of assay mixtures is run in parallelwith different test agent concentrations to obtain a differentialresponse to these concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

The screening assays provided in accordance with this invention arebased on those disclosed in International application WO 96/04557, whichis incorporated herein in its entirety. Briefly, WO 96/04557 disclosesthe use of reporter peptides that bind to active sites on targets andpossess agonist or antagonist activity at the target. These reportersare identified from recombinant libraries and are either peptides withrandom amino acid sequences or variable antibody regions with at leastone CDR region that has been randomized (rVab). The reporter peptidesmay be expressed in cell recombinant expression systems, such as forexample in E. coli, or by phage display (see WO 96/04557 and Kay et al.1996, Mol. Divers. 1(2):139-40, both of which are incorporated herein byreference). The reporters identified from the libraries may then be usedin accordance with this invention either as therapeutics themselves, orin competition binding assays to screen for other molecules, preferablysmall, active molecules, which possess similar properties to thereporters and may be developed as drug candidates to provide agonist orantagonist activity. Preferably, these small organic molecules areorally active.

The basic format of an in vitro competitive receptor binding assay asthe basis of a heterogeneous screen for small organic molecularreplacements for insulin may be as follows: occupation of the activesite of IR is quantified by time-resolved fluorometric detection (TRFD)with streptavidin-labeled europium (saEu) complexed to biotinylatedpeptides (bP). In this assay, saEu forms a ternary complex with bP andIR (i.e., IR:bP:saEu complex). The TRFD assay format iswell-established, sensitive, and quantitative (Tompkins et al., 1993, J.Immunol. Methods 163:209-216). The assay can use a single-chain antibodyor a biotinylated peptide. Furthermore, both assay formats faithfullyreport the competition of the biotinylated ligands binding to the activesite of IR by insulin.

In these assays, soluble IR is coated on the surface of microtiterwells, blocked by a solution of 0.5% bovine serum albumin (BSA) and 2%non-fat milk in PBS, and then incubated with biotinylated peptide orrVab. Unbound bP is then washed away and saEu is added to complex withreceptor-bound bP. Upon addition of the acidic enhancement solution, thebound europium is released as free Eu³⁺ which rapidly forms a highlyfluorescent and stable complex with components of the enhancementsolution. The IR:bP bound saEu is then converted into its highlyfluorescent state and detected by a detector such as Wallac Victor II(EG&G Wallac, Inc.)

Phage display libraries can also be screened for ligands that bind to IRor IGF-1R, as described above. Details of the construction and analysesof these libraries, as well as the basic procedures for biopanning andselection of binders, have been published (see, e.g., WO 96/04557;Mandecki et al., 1997, Display Technologies—Novel Targets andStrategies, P. Guttry (ed), International Business Communications, Inc.Southborogh, Mass., pp. 231-254; Ravera et al., 1998, Oncogene16:1993-1999; Scott and Smith, 1990, Science 249:386-390); Grihalde etal., 1995, Gene 166:187-195; Chen et al., 1996, Proc. Natl. Acad. Sci.USA 93:1997-2001; Kay et al., 1993, Gene 128:59-65; Carcamo et al.,1998, Proc. Natl. Acad. Sci. USA 95:11146-11151; Hoogenboom, 1997,Trends Biotechnol. 15:62-70; Rader and Barbas, 1997, Curr. Opin.Biotechnol. 8:503-508; all of which are incorporated herein byreference).

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesize or where it is unsuitable for a particularmethod of administration, e.g., peptides are generally unsuitable activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis, andtesting are generally used to avoid large-scale screening of moleculesfor a target property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. First, the particular parts ofthe compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide(e.g., by substituting each residue in turn). These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modeledaccording to its physical properties (e.g., stereochemistry, bonding,size, and/or charge), using data from a range of sources (e.g.,spectroscopic techniques, X-ray diffraction data, and NMR).Computational analysis, similarity mapping (which models the chargeand/or volume of a pharmacophore, rather than the bonding betweenatoms), and other techniques can be used in this modeling process.

In a variant of this approach, the three dimensional structure of theligand and its binding partner are modeled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

A template molecule is then selected, and chemical groups that mimic thepharmacophore can be grafted onto the template. The template moleculeand the chemical groups grafted on to it can conveniently be selected sothat the mimetic is easy to synthesize, is likely to bepharmacologically acceptable, does not degrade in vivo, and retains thebiological activity of the lead compound. The mimetics found are thenscreened to ascertain the extent they exhibit the target property, or towhat extent they inhibit it. Further optimization or modification canthen be carried out to arrive at one or more final mimetics for in vivoor clinical testing.

This invention provides specific IR and IGF-1R amino acid sequences thatfunction as either agonists or antagonists at IR and/or IGF-1R.Additional sequences may be obtained in accordance with the proceduresdescribed herein.

H. Use of the Peptides Provided by this Invention

The IR and IGF-1R agonist and antagonist peptides provided by thisinvention are useful as lead compounds for identifying other more potentor selective therapeutics, assay reagents for identifying other usefulligands by, for example, competition screening assays, as research toolsfor further analysis of IR and IGF-1R, and as potential therapeutics inpharmaceutical compositions. In one embodiment, one or more of thedisclosed peptides can be provided as components in a kit foridentifying other ligands (e.g., small, organic molecules) that bind toIR or IGF-1R. Such kits may also comprise IR or IGF-1R, or functionalfragments thereof. The peptide and receptor components of the kit may belabeled (e.g., by radioisotopes, fluorescent molecules, chemiluminescentmolecules, enzymes or other labels), or may be unlabeled and labelingreagents may be provided. The kits may also contain peripheral reagentssuch as buffers, stabilizers, etc. Instructions for use can also beprovided.

In another embodiment, the peptide sequences provided by this inventioncan be used to design secondary peptide libraries, which are derivedfrom the peptide sequences, and include members that bind to Site 1and/or Site 2 of IR or IGF-1R. Such libraries can be used to identifysequence variants that increase or otherwise modulate the binding and/oractivity of the original peptide at IR or IGF-1R, as described in therelated applications of Beasley et al. International ApplicationPCT/US00/08528, filed Mar. 29, 2000, and Beasley et al., U.S.application Ser. No. 09/538,038, filed Mar. 29, 2000, in accordance withwell-established techniques.

IR agonist amino acid sequences provided by this invention are useful asinsulin analogs and may therefore be developed as treatments fordiabetes or other diseases associated with a decreased response orproduction of insulin. For use as an insulin supplement or replacement,amino acid sequences include D117/H2C: FHENFYDWFVRQVSK (SEQ ID NO:1780);D117/H2C minus terminal lysine: FHENFYDWFVRQVS (SEQ ID NO:1557); D118:DYKDFYDAIQLVRSARAGGTRDKK (SEQ ID NO:1781); D118 minus FLAG® tag andterminal lysines: FYDAIQLVRSARAGGTRD (SEQ ID NO:1782); D119:KDRAFYNGLRDLVGAVYGAWDKK (SEQ ID NO:1733); D119 minus terminal lysines:KDRAFYNGLRDLVGAVYGAWD (residues 1-21 of SEQ ID NO:1733); D116/JBA5:DYKDLCQSWGVRIGWLAGLCPKK (SEQ ID NO:1541); D116/JBA5 minus FLAG® tag andterminal lysines: LCQSWGVRIGWLAGLCP (SEQ ID NO:1542); D113/H2:DYKDVTFTSAVFHENFYDWFVRQVSKK (SEQ ID NO:1783); D113/H2 minus FLAG® tagand terminal lysines: VTFTSAVFHENFYDWFVRQVS (SEQ ID NO:1784); and S175:GRVDWLQRNANFYDWFVAELG (SEQ ID NO:1560). Preferred peptide dimersequences are represented by S325, S332, S333, S335, S337, S353,S374-S376, S378, S379, S381, S414, S415, and S418 (see Table 7). Otherpreferred dimers sequences are represented by S455, S457, S458, S467,S468, S471, S499, S510, S518, S519, and S520 sequences (see Table 7).Especially preferred is the S519 dimer sequence, which shows in vitroand in vivo activity comparable to insulin (see FIGS. 31A-C, 32A-B, and33).

IGF-1R antagonist amino acid sequences provided by this invention areuseful as treatments for cancers, including, but not limited to, breast,prostate, colorectal, and ovarian cancers. Human and breast cancers areresponsible for over 40,000 deaths per year, as present treatments suchas surgery, chemotherapy, radiation therapy, and immunotherapy showlimited success. The IGF-1R antagonist amino acid sequences disclosedherein are also useful for the treatment or prevention of diabeticretinopathy. Recent reports have shown that a previously identifiedIGF-1R antagonist can suppress retinal neovascularization, which causesdiabetic retinopathy (Smith et al., 1999, Nat. Med. 5:1390-1395).Preferred IGF-1R antagonist amino acid sequences include thosecomprising the sequences of RP33-IGF and RP33K-IGF (Tables 24-26).

IGF-1R agonist amino acid sequences provided by this invention areuseful for development as treatments for neurological disorders,including stroke and diabetic neuropathy. Reports of several differentgroups implicate IGF-1R in the reduction of global brain ischemia, andsupport the use of IGF-1 for the treatment of diabetic neuropathy(reviewed in Auer et al., 1998, Neurology 51:S39-S43; Apfel, 1999, Am.J. Med. 107:34 S-42S). The IGF-1R agonist peptides of the invention maybe useful for enhancing the survival of cells and/or blocking apoptosisin cells. Preferred IGF-1R agonist amino acid sequences include thosecomprising the sequences of G33, RP48, RP60, and RP30-IGF-12-RP31-IGF(Tables 27-29).

I. Methods of Administration

The amino acid sequences of this invention may be administered aspharmaceutical compositions comprising standard carriers known in theart for delivering proteins and peptides and by gene therapy.Preferably, a pharmaceutical composition includes, in admixture, apharmaceutically (i.e., physiologically) acceptable carrier, excipient,or diluent, and one or more of an IR or IGF-1R agonist or antagonistpeptide, as an active ingredient. The preparation of pharmaceuticalcompositions that contain peptides as active ingredients is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions, however, solidforms suitable for solution in, or suspension in, liquid prior toinjection can also be prepared. The preparation can also be emulsified.The active therapeutic ingredient is often mixed with excipients thatare pharmaceutically (i.e., physiologically) acceptable and compatiblewith the active ingredient. Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH-buffering agents, which enhance the effectiveness of the activeingredient.

An IR or IGF-1R agonist or antagonist peptide can be formulated into apharmaceutical composition as neutralized physiologically acceptablesalt forms. Suitable salts include the acid addition salts (i.e., formedwith the free amino groups of the peptide molecule) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed from the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The pharmaceutical compositions can be administered systemically by oralor parenteral routes. Non-limiting parenteral routes of administrationinclude subcutaneous, intramuscular, intraperitoneal, intravenous,transdermal, inhalation, intranasal, intra-arterial, intrathecal,enteral, sublingual, or rectal. Due to the labile nature of the aminoacid sequences parenteral administration is preferred. Preferred modesof administration include aerosols for nasal or bronchial absorption;suspensions for intravenous, intramuscular, intrasternal orsubcutaneous, injection; and compounds for oral administration.

Intravenous administration, for example, can be performed by injectionof a unit dose. The term “unit dose” when used in reference to apharmaceutical composition of the present invention refers to physicallydiscrete units suitable as unitary dosage for humans, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent; i.e., liquid used to dilute a concentrated or pure substance(either liquid or solid), making that substance the correct (diluted)concentration for use. For injectable administration, the composition isin sterile solution or suspension or may be emulsified inpharmaceutically- and physiologically-acceptable aqueous or oleaginousvehicles, which may contain preservatives, stabilizers, and material forrendering the solution or suspension isotonic with body fluids (i.e.,blood) of the recipient.

Excipients suitable for use are water, phosphate buffered saline, pH7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, diluteethanol, and the like, and mixtures thereof. Illustrative stabilizersare polyethylene glycol, proteins, saccharides, amino acids, inorganicacids, and organic acids, which may be used either on their own or asadmixtures. The amounts or quantities, as well as routes ofadministration, used are determined on an individual basis, andcorrespond to the amounts used in similar types of applications orindications known to those of skill in the art.

Pharmaceutical compositions are administered in a manner compatible withthe dosage formulation, and in a therapeutically effective amount. Thequantity to be administered depends on the subject to be treated,capacity of the subject's immune system to utilize the activeingredient, and degree of modulation of IR or IGF-1R activity desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are specific for eachindividual. However, suitable dosages may range from about 10 to 200nmol active peptide per kilogram body weight of individual per day anddepend on the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusions sufficient to maintainpicomolar concentrations (e.g., approximately 1 pM to approximately 10nM) in the blood are contemplated. An exemplary formulation comprisesthe IR or IGF-1R agonist or antagonist peptide in a mixture with sodiumbusulfite USP (3.2 mg/ml); disodium edetate USP (0.1 mg/ml); and waterfor injection q.s.a.d. (1 ml).

Further guidance in preparing pharmaceutical formulations can be foundin, e.g., Gilman et al. (eds), 1990, Goodman and Gilman's: ThePharmacological Basis of Therapeutics, 8th ed., Pergamon Press; andRemington's Pharmaceutical Sciences, 17th ed., 1990, Mack PublishingCo., Easton, Pa.; Avis et al. (eds), 1993, Pharmaceutical Dosage Forms:Parenteral Medications, Dekker, New York; Lieberman et al. (eds), 1990,Pharmaceutical Dosage Forms: Disperse Systems, Dekker, N.Y.

The present invention further contemplates compositions comprising an IRor IGF-1R agonist or antagonist peptide, and a physiologicallyacceptable carrier, excipient, or diluent as described in detail herein.

The constructs as described herein may also be used in gene transfer andgene therapy methods to allow the expression of one or more amino acidsequences of the present invention. The amino acid sequences of thepresent invention can be used for gene therapy and thereby provide analternative method of treating diabetes which does not rely on theadministration or expression of insulin. Expressing insulin for use ingene therapy requires the expression of a precursor product, which mustthen undergo processing including cleavage and disulfide bond formationto form the active product. The amino acid sequences of this invention,which possess activity, are relatively small, and thus do not requirethe complex processing steps to become active. Accordingly, thesesequences provide a more suitable product for gene therapy.

Gene transfer systems known in the art may be useful in the practice ofthe gene therapy methods of the present invention. These include viraland non-viral transfer methods. A number of viruses have been used asgene transfer vectors, including polyoma, i.e., SV40 (Madzak et al.,1992, J. Gen. Virol., 73:1533-1536), adenovirus (Berkner, 1992, Curr.Top. Microbiol. Immunol., 158:39-6; Berkner et al., 1988, BioTechniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412;Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584;Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl.Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. GeneTher., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology,24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top.Microbiol. Immunol. 158:91-123; Ohi et al., 1990, Gene, 89:279-282),herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top.Microbiol. Immunol. 158:67-90; Johnson et al., 1992, J. Virol.,66:2952-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem.Pharmacol. 40:2189-2199), and retroviruses of avian (Brandyopadhyay etal., 1984, Mol. Cell. Biol., 4:749-754; Petropouplos et al., 1992, J.Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol.Immunol. 158:1-24; Miller et al., 1985, Mol. Cell. Biol., 5:431-437;Sorge et al., 1984, Mol. Cell. Biol., 4:1730-1737; Mann et al., 1985, J.Virol., 54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Mosthuman gene therapy protocols have been based on disabled murineretroviruses.

Non-viral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham et al.,1973, Virology, 52:456-467; Pellicer et al., 1980, Science,209:1414-1422), mechanical techniques, for example microinjection(Anderson et al., 1980, Proc. Natl. Acad. Sci. USA, 77:5399-5403; Gordonet al., 1980, Proc. Natl. Acad. Sci. USA, 77:7380-7384; Brinster et al.,1981, Cell, 27:223-231; Constantini et al., 1981, Nature, 294:92-94),membrane fusion-mediated transfer via liposomes (Felgner et al., 1987,Proc. Natl. Acad. Sci. USA, 84:7413-7417; Wang et al., 1989,Biochemistry, 28:9508-9514; Kaneda et al., 1989, J. Biol. Chem.,264:12126-12129; Stewart et al., 1992, Hum. Gene Ther. 3:267-275; Nabelet al., 1990, Science, 249:1285-1288; Lim et al., 1992, Circulation,83:2007-2011; U.S. Pat. Nos. 5,283,185 and 5,795,587), and direct DNAuptake and receptor-mediated DNA transfer (Wolff et al., 1990, Science,247:1465-1468; Wu et al., 1991, BioTechniques, 11:474-485; Zenke et al.,1990, Proc. Natl. Acad. Sci. USA, 87:3655-3659; Wu et al., 1989, J.Biol. Chem., 264:16985-16987; Wolff et al., 1991, BioTechniques,11:474-485; Wagner et al., 1991, Proc. Natl. Acad. Sci. USA,88:4255-4259; Cotten et al., 1990, Proc. Natl. Acad. Sci. USA,87:4033-4037; Curiel et al., 1991, Proc. Natl. Acad. Sci. USA,88:8850-8854; Curiel et al., 1991, Hum. Gene Ther. 3:147-154).

Many types of cells and cell lines (e.g., primary cell lines orestablished cell lines) and tissues are capable of being stablytransfected by or receiving the constructs of the invention. Examples ofcells that may be used include, but are not limited to, stem cells, Blymphocytes, T lymphocytes, macrophages, other white blood lymphocytes(e.g., myelocytes, macrophages, or monocytes), immune system cells ofdifferent developmental stages, erythroid lineage cells, pancreaticcells, lung cells, muscle cells, liver cells, fat cells, neuronal cells,glial cells, other brain cells, transformed cells of various celllineages corresponding to normal cell counterparts (e.g., K562, HEL,HL60, and MEL cells), and established or otherwise transformed cellslines derived from all of the foregoing. In addition, the constructs ofthe present invention may be transferred by various means directly intotissues, where they would stably integrate into the cells comprising thetissues. Further, the constructs containing the DNA sequences of thepeptides of the invention can be introduced into primary cells atvarious stages of development, including the embryonic and fetal stages,so as to effect gene therapy at early stages of development.

In one approach, plasmid DNA is complexed with a polylysine-conjugatedantibody specific to the adenovirus hexon protein, and the resultingcomplex is bound to an adenovirus vector. The trimolecular complex isthen used to infect cells. The adenovirus vector permits efficientbinding, internalization, and degradation of the endosome before thecoupled DNA is damaged.

In another approach, liposome/DNA is used to mediate direct in vivo genetransfer. While in standard liposome preparations the gene transferprocess is non-specific, localized in vivo uptake and expression havebeen reported in tumor deposits, for example, following direct in situadministration (Nabel, 1992, Hum. Gene Ther. 3:399-410).

Suitable gene transfer vectors possess a promoter sequence, preferably apromoter that is cell-specific and placed upstream of the sequence to beexpressed. The vectors may also contain, optionally, one or moreexpressible marker genes for expression as an indication of successfultransfection and expression of the nucleic acid sequences contained inthe vector. In addition, vectors can be optimized to minimize undesiredimmunogenicity and maximize long-term expression of the desired geneproduct(s) (see Nabe, 1999, Proc. Natl. Acad. Sci. USA 96:324-326).Moreover, vectors can be chosen based on cell-type that is targeted fortreatment.

Illustrative examples of vehicles or vector constructs for transfectionor infection of the host cells include replication-defective viralvectors, DNA virus or RNA virus (retrovirus) vectors, such asadenovirus, herpes simplex virus and adeno-associated viral vectors.Adeno-associated virus vectors are single stranded and allow theefficient delivery of multiple copies of nucleic acid to the cell'snucleus. Preferred are adenovirus vectors. The vectors will normally besubstantially free of any prokaryotic DNA and may comprise a number ofdifferent functional nucleic acid sequences. An example of suchfunctional sequences may be a DNA region comprising transcriptional andtranslational initiation and termination regulatory sequences, includingpromoters (e.g., strong promoters, inducible promoters, and the like)and enhancers which are active in the host cells. Also included as partof the functional sequences is an open reading frame (polynucleotidesequence) encoding a protein of interest. Flanking sequences may also beincluded for site-directed integration. In some situations, the5′-flanking sequence will allow homologous recombination, thus changingthe nature of the transcriptional initiation region, so as to providefor inducible or non-inducible transcription to increase or decrease thelevel of transcription, as an example.

In general, the encoded and expressed peptide may be intracellular,i.e., retained in the cytoplasm, nucleus, or in an organelle, or may besecreted by the cell. For secretion, a signal sequence may be fused tothe peptide sequence. As previously mentioned, a marker may be presentfor selection of cells containing the vector construct. The marker maybe an inducible or non-inducible gene and will generally allow forpositive selection under induction, or without induction, respectively.Examples of marker genes include neomycin, dihydrofolate reductase,glutamine synthetase, and the like. The vector employed will generallyalso include an origin of replication and other genes that are necessaryfor replication in the host cells, as routinely employed by those havingskill in the art. As an example, the replication system comprising theorigin of replication and any proteins associated with replicationencoded by a particular virus may be included as part of the construct.The replication system must be selected so that the genes encodingproducts necessary for replication do not ultimately transform thecells. Such replication systems are represented by replication-defectiveadenovirus (see G. Acsadi et al., 1994, Hum. Mol. Genet. 3:579-584) andby Epstein-Barr virus. Examples of replication defective vectors,particularly, retroviral vectors that are replication defective, areBAG, (see Price et al., 1987, Proc. Natl. Acad. Sci. USA, 84:156; Saneset al., 1986, EMBO J., 5:3133). It will be understood that the finalgene construct may contain one or more genes of interest, for example, agene encoding a bioactive metabolic molecule. In addition, cDNA,synthetically produced DNA or chromosomal DNA may be employed utilizingmethods and protocols known and practiced by those having skill in theart.

According to one approach for gene therapy, a vector encoding an IR orIGF-1R agonist or antagonist peptide is directly injected into therecipient cells (in vivo gene therapy). Alternatively, cells from theintended recipients are explanted, genetically modified to encode an IRor IGF-1R agonist or antagonist peptide, and reimplanted into the donor(ex vivo gene therapy). An ex vivo approach provides the advantage ofefficient viral gene transfer, which is superior to in vivo genetransfer approaches. In accordance with ex vivo gene therapy, the hostcells are first transfected with engineered vectors containing at leastone gene encoding an IR or IGF-1R agonist or antagonist peptide,suspended in a physiologically acceptable carrier or excipient such assaline or phosphate buffered saline, and the like, and then administeredto the host or host cells. The desired gene product is expressed by theinjected cells, which thus introduce the gene product into the host. Theintroduced gene products can thereby be utilized to treat or amelioratea disorder that is related to altered insulin or IGF-1 levels (e.g.,diabetes).

The described constructs may be administered in the form of apharmaceutical preparation or composition containing a pharmaceuticallyacceptable carrier and a physiological excipient, in which preparationthe vector may be a viral vector construct, or the like, to target thecells, tissues, or organs of the recipient organism of interest,including human and non-human mammals. The composition may be formed bydispersing the components in a suitable pharmaceutically acceptableliquid or solution such as sterile physiological saline or otherinjectable aqueous liquids. The amounts of the components to be used insuch compositions may be routinely determined by those having skill inthe art. The compositions may be administered by parenteral routes ofinjection, including subcutaneous, intravenous, intramuscular, andintrasternal.

J. Cancer Therapeutics

In recent experiments, embryo fibroblasts from IGF-1R knock-out micehave been shown to be highly resistant to transformation by oncogenessuch as SV40 T antigen, activated Ha-ras, activated Src, and others (B.Valentinis and R. Baserga, 2001, Mol. Pathol., 54:133-137). Thissuggested that IGF-1R was required to mediate malignant transformationby these oncogenes. In addition, IGF-1 and IGF-1R have been shown to actas transforming factors in various forms of human cancer (see above).IGF-1 and IGF-2 have also been implicated as factors in the malignanttransformation of several tissues. Transgenic mice that express atruncated form of IGF-1 that has a decreased affinity for IGFBPs(des(1-3) IGF-11), show increased incidence of mammary tumors (Hadsellet al., 2000, Oncogene 19:889-898). In addition, mice over-expressingIGF-1I in mammary glands showed increased mammary tumor formation (Bateset al., 1995, Br. J. Cancer 72:1189-1193). Transgenic mice thatoverexpress IGF-1 in the basal layer of the skin show hyperplasia of theepidermis and increased promotion of spontaneous tumors (DiGiovanni etal., 2000, Cancer Res. 60:1561-1570).

IGF-1R also appears to cross-talk with other hormone receptors.Considerable evidence suggests that estrogen can act to increaseexpression of IGF-1R. This is of particular importance in breast cancer,where the expression of IGF-1R correlates with expression of theestrogen receptor (ER). IGF-1R expression is higher in tumors from ERpositive patients. Accordingly, IGF-1R expression could be used as aprognostic marker for breast cancer patients. In addition, high levelsof IRS-1, a key intermediate in the IGF-1R signal transduction cascade,correlates with tumor size and shorter disease-free survival in patientswith ER positive tumors (D. Sachdev and D. Yee, 2001, Endocr. Relat.Cancer 8:197-209). In addition, treatment with anti-estrogens has beenshown to decrease the expression of IGF-1R and IRS-1 (Chan et al., 2001,Clin. Cancer Res. 7:2545-2554). Thus, the cross-talk between IGF-1R andER may be complex. Yet, it is clear that IGF-signaling promotesmalignant transformation in mammary glands. Interestingly, ER positiveMCF-7 cells treated with IGF-1 show a sustained activation of thePI3K-Akt pathway and protection against apoptosis induced by serumdeprivation. In contrast, ER negative MDA-MB 231 cells show only atransient activation of PI3K-Akt pathway (Bartucci et al., 2001, CancerRes. 61:6747-6754).

Studies have also revealed a connection between IGF-1R-mediatedsignaling and epidermal growth factor (EGF)-induced signaling throughErbB-receptors. IGF-1R and ErbB-2 (Neu/Her2) have been observed to formhetero-oligomers induced by stimulation with heregulin or IGF-1 (Balanaet al., 2001, Oncogene, 19:34-47, 2001). In glioblastomas, resistance toa chemical inhibitor of the ErbB receptor tyrosine kinase has beencorrelated with increased IGF-1R expression and constitutive PI3Ksignaling (Chakravarti et al., 2002, Cancer Res. 62:200-207). In breastcancer cell lines over expressing ErbB-2, increased IGF-1R signaling wasobserved in the presence of the anti-ErbB-2 receptor monoclonal antibodyHerceptin®/trastuzumab (Lu et al., 2001, J. Natl. Cancer Inst.93:1852-1857).

Modulation of IGF-signaling in various malignant cells has providedfurther evidence for the involvement of the IGF-1R in cancer. Abrogationof IGF-1R expression by antisense RNA reversed the transformed phenotypein cervical cancer cells. Antisense to IGF-1R also inhibitedglioblastoma and melanoma xenografts in nude mice (Resnicoff et al.,1994, Cancer Res. 54:4848-4850; Resnicoff et al., 1994, Cancer Res.54:2218-2222; Nakamura et al., 2000, Cancer Res. 60:760-765, 2000).Experiments have also indicated that IGF-1R is involved in thedevelopment and maintenance of metastatic phenotypes. In particular,high expression of a dominant negative mutant of IGF-1R (486stop) in ERpositive breast cancer cells has been shown to inhibit adhesion,invasion, and metastasis of the cells (Dunn et al., 1998, Cancer Res.58:3353-3361). Moreover, lung carcinoma cells exhibited an enhancedmetastatic phenotype following overexpression of IGF-1R (Long et al.,1998, Exp. Cell Res. 238:116-121). In addition, activation of IGF-1R hasbeen shown to block apoptotic pathways. Apoptosis in mammary glands wasinhibited in IGF-1 transgenic mice (Hadsell et al., 2000, Oncogene19:889-898). Moreover, down-regulation of IGF-1R function, either byantisense strategies or dominant negative mutants, caused massiveapoptosis of tumor cells in vitro and in vivo. IGF-1 has also been shownto inhibit apoptosis associated with transformation by the c-myconcogene and apoptosis induced by chemotherapeutic agents. Theanti-apoptotic signaling of IGF-1 has been attributed to the PI3K-Aktpathway, although other pathways may mediate similar effects (Butt etal., 1999, Immunol. Cell Biol. 77:256-262; B. Valentinis and R. Baserga,2001, Mol. Pathol. 54:133-137).

The sum of these observations indicate the importance of identifyingantagonists or inhibitors of IGF-1 and/or IGF-1R. Attempts have beenmade to develop clinically relevant inhibitors of IGF-1R usingmonoclonal antibodies, antisense strategies, and peptide fragmentsderived from the natural ligand (Dunn et al., 1998, Cancer Res.58:3353-3361; Z. Pietrzkowski et al., 1992, Cancer Res. 52:6447-6451; Z.Pietrzkowski et al., 1993, Cancer Res. 53:1102-1106; Rubini et al.,1999, Exp. Cell Res. 251:22-32). Using an alternate approach, thisinvention provides methods, kits, and compositions (e.g., pharmaceuticalcompositions) comprising IGF-1R antagonist peptides, or small moleculemimetics thereof, that can be useful in the diagnosis, treatment, andmonitoring of one or more cancers. In some cases, the compositions,methods, and kits of the invention can also be used to determine theprognosis of a IGF-related medical condition (e.g., cancer).Advantageously, certain IGF-1R antagonist peptides disclosed herein arespecific for Site 1 or Site 2 of the IGF-1 receptor.

In accordance with the invention, non-limiting cancer types includecarcinoma, sarcoma, myeloma, leukemia, and lymphoma, and mixed types ofcancers, such as adenosquamous carcinoma, mixed mesodermal tumor,carcinosarcoma, and teratocarcinoma. Representative cancers include, butare not limited to, bladder cancer, lung cancer, breast cancer, coloncancer, rectal cancer, endometrial cancer, ovarian cancer, head and neckcancer, prostate cancer, and melanoma. Specifically included areAIDS-related cancers (e.g., Kaposi's Sarcoma, AIDS-related lymphoma),bone cancers (e.g., osteosarcoma, malignant fibrous histiocytoma ofbone, Ewing's Sarcoma, and related cancers), and hematologic/bloodcancers (e.g., adult acute lymphoblastic leukemia, childhood acutelymphoblastic leukemia, adult acute myeloid leukemia, childhood acutemyeloid leukemia, chronic lymphocytic leukemia, chronic myelogenousleukemia, hairy cell leukemia, cutaneous T-cell lymphoma, adultHodgkin's disease, childhood Hodgkin's disease, Hodgkin's disease duringpregnancy, mycosis fungoides, adult non-Hodgkin's lymphoma, childhoodnon-Hodgkin's lymphoma, non-Hodgkin's lymphoma during pregnancy, primarycentral nervous system lymphoma, Sezary syndrome, cutaneous T-celllymphoma, Waldenström's macroglobulinemia, multiple myeloma/plasma cellneoplasm, myelodysplastic syndrome, and myeloproliferative disorders).

Also included are brain cancers (e.g., adult brain tumor, childhoodbrain stem glioma, childhood cerebellar astrocytoma, childhood cerebralastrocytoma, childhood ependymoma, childhood medulloblastoma,supratentorial primitive neuroectodermal and pineal, and childhoodvisual pathway and hypothalamic glioma), digestive/gastrointestinalcancers (e.g., anal cancer, extrahepatic bile duct cancer,gastrointestinal carcinoid tumor, colon cancer, esophageal cancer,gallbladder cancer, adult primary liver cancer, childhood liver cancer,pancreatic cancer, rectal cancer, small intestine cancer, and gastriccancer), musculoskeletal cancers (e.g., childhood rhabdomyosarcoma,adult soft tissue sarcoma, childhood soft tissue sarcoma, and uterinesarcoma), and endocrine cancers (e.g., adrenocortical carcinoma,gastrointestinal carcinoid tumor, islet cell carcinoma (endocrinepancreas), parathyroid cancer, pheochromocytoma, pituitary tumor, andthyroid cancer).

Further included are neurologic cancers (e.g., neuroblastoma, pituitarytumor, and primary central nervous system lymphoma), eye cancers (e.g.,intraocular melanoma and retinoblastoma), genitourinary cancers (e.g.,bladder cancer, kidney (renal cell) cancer, penile cancer, transitionalcell renal pelvis and ureter cancer, testicular cancer, urethral cancer,Wilms' tumor and other childhood kidney tumors), respiratory/thoraciccancers (e.g., non-small cell lung cancer, small cell lung cancer,malignant mesothelioma, and malignant thymoma), germ cell cancers (e.g.,childhood extracranial germ cell tumor and extragonadal germ celltumor), skin cancers (e.g., melanoma, and merkel cell carcinoma),gynecologic cancers (e.g., cervical cancer, endometrial cancer,gestational trophoblastic tumor, ovarian epithelial cancer, ovarian germcell tumor, ovarian low malignant potential tumor, uterine sarcoma,vaginal cancer, and vulvar cancer), and unknown primary cancers.

Specific breast cancers include, but are not limited to, non-invasivecancers, such as ductal carcinoma in situ (DCIS), intraductal carcinomalobular carcinoma in situ (LCIS), papillary carcinoma, andcomedocarcinoma, or invasive cancers, such as adenocarcinomas, orcarcinomas, e.g., infiltrating ductal carcinoma, infiltrating lobularcarcinoma, infiltrating ductal and lobular carcinoma, medullarycarcinoma, mucinous (colloid) carcinoma, comedocarcinoma, Paget'sDisease, papillary carcinoma, tubular carcinoma, and inflammatorycarcinoma. Specific prostate cancers may include adenocarcinomas andsarcomas, or pre-cancerous conditions, such as prostate intraepithelialneoplasia (PIN). Specific lung cancers include those relating to tumorssuch as bronchial carcinoid (bronchial adenoma), chondromatous hamartoma(benign), solitary lymphoma, and sarcoma (malignant) tumors, as well aslung cancers relating to multifocal lymphomas. Bronchogenic carcinomasmay present as squamous cell carcinomas, small cell carcinomas,non-small cell carcinomas, or adenocarcinomas.

The IGF-1R antagonist peptides of the invention may be administeredindividually, or in combination with other IGF-1 or IGF-1R antagonistsor inhibitors. Alternatively, the disclosed IGF-1R antagonist peptidescan be used in combination with other cancer therapies, e.g., surgery,radiation, biological response modification, immunotherapy, hormonetherapy, and/or chemotherapy. For prostate cancers, non-limitingexamples of chemotherapeutic agents include docetaxel, paclitaxel,estramustine, etoposide, vinblastine, mitoxantrone, and paclitaxel. Forbreast cancers, non-limiting examples of chemotherapeutic and biologicalagents include cyclophosphamide, methotrexate, 5-fluorouracil,doxorubicin, tamoxifen, paclitaxel, docetaxel, navelbine, capecitabine,mitomycin C, Interferons, interleukin-2, lymphocyte-activated killercells, tumor necrosis factors, and monoclonal antibodies (e.g., mAb toHER-2/neu receptor (trastuzumab) Herceptin®). For lung cancers,non-limiting examples of chemotherapeutic and biological agents include,but are not limited to, platinum compounds (e.g., cisplatin orcarboplatin), vinca alkaloids (e.g., vinorelbine, vincristine, orvinblastine), taxines (e.g., docetaxel or paclitaxel), and varioustopoisomerase inhibitors.

EXAMPLES

The examples as set forth herein are meant to exemplify the variousaspects of the present invention and are not intended to limit theinvention in any way.

The following materials were used in the examples described below.Soluble IGF-1R was obtained from R&D Systems (Minneapolis, Minn.; Cat. #391-GR/CF). Insulin receptor was prepared according to Bass et al.,1996. The insulin was either from Sigma (St. Louis, Mo.; Cat. # 1-0259)or Boehringer. The IGF-1 was from PeproTech (Cat. # 100-11). Allsynthetic peptides were synthesized by Novo Nordisk, AnaSpec, Inc. (SanJose, Calif.), PeptioGenics (Livermore, Calif.), or Research Genetics(Huntsville, Ala.) at >80% purity. The Maxisorb Plates were from NUNCvia Fisher (Cat. # 12565347). The HRP/Anti-M13 conjugate was fromPharmacia (Cat. # 27-9421-01). The ABTS solution was from BioF/X (Cat. #ABTS-0100-04).

Example 1 Monomer and Dimer Peptides

A. Cloning

Monomer and dimer peptides were constructed and expressed as proteinfusions to a chitin binding domain (CBD) using the pTYB2 vector from theIMPACT™-CN system (New England Biolabs (NEB), Beverly, Mass.). The pTYB2vector encodes a protein-splicing element (termed intein), whichinitiates self-cleavage upon the addition of DTT. The inteinself-cleavage separates the dimer from the affinity tag, to allowpurification.

In the pTYB2 construct, the C-terminus of the peptide sequence was fusedto the N-terminus of the intein/CBD sequence. Two peptide-flankingepitope tags were included: a shortened-FLAG® at the N-terminus andE-Tag at the C-terminus. This fusion was generated by ligating a vectorfragment encoding the intein/CBD with a PCR product encoding the peptideof interest.

The vector fragment was obtained by digesting at appropriate restrictionsites the pTBY2 vector. The digested DNA fragment was resolved on a 1%agarose gel, excised, and purified by QIAEXII (QIAGEN, Valencia,Calif.). To obtain the PCR product of the target proteins, primers weresynthesized which anneal to appropriate sequences. The vector and insertwere ligated overnight at 15° C. The ligation product was purified usingQIAquick spin columns (QIAGEN) and electroporations were performed at1500 V in an electroporation cuvette (0.1 mm gap; 0.5 ml volume)containing 10 ng of DNA and 40 μl of E. coli strain BL21.

Immediately following electroporation, 1 ml of pre-warmed (40° C.) 2xYTmedium containing 2% glucose (2xYT-G) was added to the transformants.The transformants were grown at 37° C. for 1 h, and then plated onto2xYT-AG plates and incubated overnight at 37° C. Individual colonieswere isolated and used to innoculate 2xYT-G. The cultures were grownovernight at 37° C. Plasmid DNA was isolated from the cultures andsequencing was performed to confirm that the correct construct wasobtained.

B. Small-Scale Expression of Peptide-CBD Fusion Proteins

E. coli ER2566 (New England Biolabs) containing plasmids encodingpeptide-CBD fusion proteins were grown in 2xYT-AG at 37° C. overnight,with agitation (250 rpm). The following day, the cultures were used toinoculate media (2x YT-G) to obtain an OD₆₀₀ of 0.1. Upon reaching anOD₆₀₀ of 0.6, expression of the fusion protein was induced by theaddition of IPTG (isopropyl-β-D-thiogalactopyranoside) to a finalconcentration of 0.3 mM. Cells were grown for 3 h. Following this, cellswere pelleted by centrifugation and the cell pellets were analyzed bySDS-PAGE electrophoresis. Production of the correct molecular weightfusion proteins was confirmed by Western blot analysis using themonoclonal antibody anti-E-Tag-HRP conjugate (Amersham Pharmacia).

C. Large-Scale Expression and Purification of Soluble Peptide-CBD FusionProteins

E. coli ER2566 carrying plasmids encoding the fusion proteins were grownin 2xYT-AG media at 37° C. for 8 h, with agitation (250 rpm). Thecultures were back-diluted into to 2 L volumes of 2xYT-A to achieve anOD₆₀₀ of 0.1. Upon reaching an OD₆₀₀ of 0.5, IPTG was added to a finalconcentration of 0.3 mM. Cells were grown at 30° C. overnight. The nextday cells were isolated by centrifugation. Samples of the cell pelletwere analyzed by SDS-PAGE followed by the Western blot analysis usingthe mouse monoclonal antibody anti-E-Tag-HRP conjugate (Pharmacia) tovisualize the expressed product.

D. Purification

The cell pellets were disrupted mechanically by sonication or chemicallyby treatment with the mild detergent. After removal of cell debris bycentrifugation, the soluble proteins in the clarified lysate wereprepared for chromatographic purification by dilution or dialysis intothe appropriate starting buffer. The CBD fusions were purified by chitinaffinity chromatography according to the manufacturer's instructions(New England Biolabs). The lysate was loaded onto a chitin affinitycolumn and the column was washed with 10 volumes of column buffer. Threebed volumes of the DTT containing cleavage buffer were loaded onto thecolumn and the column was incubated overnight. The next day, the targetprotein was eluted by continuing the flow of the cleavage buffer withoutDTT. The purified proteins were analyzed for purity and integrity bySDS-PAGE and Western blot analysis according to standard protocols.

Example 2 PEG-Based Dimer Peptides

A. Synthesis of the Aldehyde Containing Peptide

The peptide was synthesized by stepwise solid phase synthesis on Rinkamide Tentagel (0.21 mmol/g). Three equivalents of Fmoc-amino acids wereused. The serine residue was introduced into the peptide by eithercoupling Fmoc-Ser(tBu)-OH to the N-terminal peptide or couplingBoc-Ser(tBu) to a selectively protected lysine side-chain. The peptidewas then deprotected and cleaved from the resin by treatment with 95%TFA (trifluoroacetic acid; aq) containing TIS (triisopropylsilan).Periodate oxidation, using 2 equivalent of NalO₄ in 20% DMSO (dimethylsulfoxide)-80% phosphate buffer pH 7.5 (45 μl/μmol peptide) for 5 min atroom temperature (RT), converted the 2-amino alcohol moiety in anα-oxoacyl group. The peptide was purified immediately followingoxidation.

B. Synthesis of the PEG-Based Dimer

The unprotected and oxidized peptide (4.2 equivalent) was dimerized onthe dioxyamino-PEG (polyethylene glycol)-linker (1 equivalent) in 90%DMSO-10% 20 mM NaOAc buffer, pH 5.1 (4.2 μl/μmol peptide). The solutionwas left for 1 h at 38° C. and the progress of the reaction wasmonitored by MALDI-MS (matrix-assisted laser desorption/ionization massspectrometry). Following this, the crude dimer was purified bysemi-preparative HPLC (high performance liquid chromatography).

The molecular weights and inter peptide distance of various linkers isshown in Table 3, below.

TABLE 3 Structure Number MW MW (-2H₂O)

1 100.1 64.1

2 58.04 22.04

3 149.15 113.15

4 150.14 114.14

5 134.13 98.13

6 134.13 98.13

7 134.13 98.13

8 234.25 198.25

9 302.3 266.3

10 72.06 36.06

11 86.09 50.09

12 114.14 78.14

13 128.08 92.08

14 142.19 106.19 (HCO)4-(Lys)2-Lys- 15 Gly-NH₂

16 136.2 100.2

17 180.2 144.2

18 224.3 188.3

19 268.3 232.3

20 312.4 276.4

21 278.4 242.4

22 240.3 204.3

23 240.3 204.3

24 210.2 192.2

Example 3 Determination of Insulin Receptor Binding

IR was incubated with ¹²⁵I-labeled insulin at various concentrations oftest substance and the K_(d) was calculated. According to this method,human insulin receptor (HIR) or human IGF-1 receptor (HIGF-1R) waspurified from transfected cells after solubilization with Triton X-100.The assay buffer contained 100 mM HEPES (pH 7.8), 100 mM NaCl, 10 mMMgCl₂, 0.5% human serum albumin (HSA), 0.2% gammaglobulin and 0.025%Triton X-100. The receptor concentration was chosen to give 30-60%binding of 2000 cpm (3 pM) of its ¹²⁵I-labeled ligand (TyrA14-¹²⁵I-HI orTyr31-¹²⁵I-IGF1) and a dilution series of the substance to be tested wasadded. After equilibration for 2 days at 4° C., each sample (200 μl) wasprecipitated by addition of 400 μl 25% PEG 6000, centrifuged, washedwith 1 ml 15% PEG 6000, and counted in a gamma-counter.

The insulin/IGF-1 competition curve was fitted to a one-site bindingmodel and the calculated parameters for receptor concentration, insulinaffinity, and non-specific binding were used in calculating the bindingconstants of the test substances. Representative curves for insulincompetition are shown in FIGS. 10A-10C; 11A-11D. Qualitative data areprovided in Table 4, below.

Table 4 illustrates IR affinities for the RP9 monomer peptide andvarious RP9 monomer truncations. The results demonstrate that RP9N-terminal sequence (GSLD; SEQ ID NO:1785) and C-terminal sequence(LGKK; SEQ ID NO:1786) can be deleted without substantially affectingHIR binding affinity (Table 4).

TABLE 4 SEQ ID Site HIR Peptide NO: Formula IR Sequence Kd (mol/l) S3861559 1 1 GSLDESFYDWFERQLG 3.2 * 10⁻⁷ S395 1787 1 1 GSLDESFYDWFERQL 9.1 *10⁻⁸ S394 1788 1 1 GSLDESFYDWFERQ 8.1 * 10⁻⁸ S396 1789 1 1 GSLDESFYDWFER >2 * 10⁻⁵ S399 1790 1 1 ESFYDWFERQL 9.1 * 10⁻⁸ S400 1791 1 1 ESFYDWFERQ6.3 * 10⁻⁷

FIGS. 10A-10C demonstrate that Site 1-Site 2 heterodimer peptides 537,538, and 539 bound to IR with substantially higher (several orders ofmagnitude) affinity than corresponding monomer (D117 and 540) andhomodimer (521 and 535) peptides. FIGS. 11A-11D demonstrate that Site1-Site 2 heterodimer peptides, 537 and 538, bound to IR with markedlyhigher affinity than the monomer peptide D117.

Example 4 Adipocyte Assay for Determination of Insulin Agonist Activity

Insulin increases uptake of ³H glucose into adipocytes and itsconversion into lipid. Incorporation of ³H into the lipid phase wasdetermined by partitioning of lipid phase into a scintillant mixture,which excludes water-soluble ³H products. The effect of compounds on theincorporation of ³H glucose at a sub-maximal insulin dose wasdetermined, and the results expressed as increase relative to fullinsulin response. The method was adapted from Moody et al., 1974, Horm.Metab. Res. 6(1):12-6.

Mouse epididymal fat pads were dissected out, minced into digestionbuffer (Krebs-Ringer 25 mM HEPES, 4% HSA, 1.1 mM glucose, 0.4 mg/mlCollagenase Type 1, pH 7.4), and digested for up to 1.5 h at 36.5° C.After filtration, washing (Krebs-Ringer HEPES, 1% HSA), and resuspensionin assay buffer (Krebs-Ringer HEPES, 1% HSA), free fat cells werepipetted into 96-well Picoplates (Packard), containing test solution andapproximately an ED₂₀ insulin.

The assay was started by addition of ³H glucose (Amersham TRK 239), in afinal concentration of 0.45 mM glucose. The assay was incubated for 2 h,36.5° C., in a Labshaker incubation tower, 400 rpm, then terminated bythe addition of Permablend/Toluene scintillant (or equivalent), and theplates sealed, before standing for at least 1 h and detection in aPackard Top Counter or equivalent. A full insulin standard curve (8dose) was run as control on each plate.

Data are presented graphically, as effect of compound on an(approximate) ED₂₀ insulin response, with data normalized to a fullinsulin response. The assay can also be run at basal or maximal insulinconcentration. Representative dose-response curves for insulin and IGF-1are shown in FIGS. 12-18. Qualitative data are shown in Tables 5-7.

In free fat cell (FFC) assays, truncated synthetic RP9 monomer peptidesS390 and S394 showed potency similar to full-length RP9 monomer peptides(FIGS. 12A-12D). Truncated synthetic RP9 homodimer peptides S415 andS417 were highly potent in FFC assays, but less potent than full-lengthRP9 homodimer peptides (FIGS. 13A-13C; compare to peptides 521 and 535,described below). The potency of recombinant RP9 homodimer peptides 521and 535 in FFC assays is shown in FIGS. 14A-14C. The curves areflattened, suggesting that the binding mechanism may not be mediated bysimple intramolecular binding (FIGS. 14A-14C).

Results further indicated that synthetic RP9 homodimer peptides S337 andS374 showed increased HIR biding affinity and increased potency in FFCassays compared to synthetic RP9 monomer, S371 (Table 5). Similarly,synthetic RP9 homodimer peptides S314 and S317 showed increased HIRbinding affinity and increased potency in FFC assays compared tosynthetic RP9 monomer, S371, and various RP9 truncations (Table 6).

TABLE 5 SEQ ID Site Monomer HIR k_(d) Pep. NO: Formula IR or DimerSequence (mol/l) FFC S371 1558 1 1 M (RP9) GSLDESFYDWFERQLGKK6.3.*10⁻⁷ + S337 1792 1-1 1-1 D, C-Term 23 (GSLDESFYDWFERQLGKK-Lig)₂-231.1*10⁻⁸ +++++ S374 1793 1-1 1-1 D, N-Term 17 17-(GSLDESFYDWFERQLGKK)₂₂1.8*10⁻⁷ ++++ M = monomer; D = dimer; C-Term = C-terminal linker (C-C);N-Term = N-terminal linker (N-N); 23 and 17 represent specific chemicallinkers (see Table 3); For FEC: 0 is no effect, + is agonist, − isantagonist.

TABLE 6 SEQ ID Site Mon. or HIR K_(d) Peptide NO: Form. IR DimerSequence (mol/l) FFC S371 1558 1 1 M GSLDESFYDWFERQLGKK 6.3*10⁻⁷ + (RP9)S395 1787 1 1 M GSLDESFYDWFERQL 9.1*10⁻⁸ + S394 1788 1 1 MGSLDESFYDWFERQ 8.1*10⁻⁸ ++ S396 1789 1 1 M GSLDESFYDWFER  >2*10⁻⁵ 0 S3901794 1 1 M ESFYDWFERQLG 6.2*10⁻⁷ + S399 1790 1 1 M ESFYDWFERQL 9.1*10⁻⁸++ S400 1791 1 1 M ESFYDWFERQ 6.3*10⁻⁷ 0 S415 1795 1-1 1-1 D; C-Term(ESFYDWFERQLGK)₂-23 1.0*10⁻⁷ ++++ S417 1796 1-1 1-1 D; N-Term23-(ESFYDWFERQLG)₂ 9.2*10⁻⁷ +++ M = monomer; D = dimer; C-Term= C-terminal linker (C-C); N-Term = N-terminal linker (N-N); 23represents a specific chemical linker (see Table 3); For FEC: 0 is noeffect, + is agonist, − is antagonist; Form. = formula; Mon. = monomer;

Site 1-Site 2 dimer peptides 537 and 538 were inactive in the FFC assaysusing the standard concentration of insulin (FIGS. 15A-15C). However,Site 1-Site 2 dimer peptides 537 and 538 were antagonists in the FFCassay in the presence of a stimulating concentration of insulin (FIGS.16A-16C). In contrast, Site 2-Site 1 dimer peptide 539 was a fullagonist in the FFC assay, with a slope similar to that of insulin (FIGS.17A-17B).

Additional experiments confirmed that FFC assay activity of Site 1-Site2 dimer peptides was affected by the orientation of the monomer subunits(FIGS. 18A-18D). In particular, dimer peptides comprising Site 1 (S372or S373) and Site 2 (S451 or S452) monomer subunits exhibited antagonistactivity in the Site 1-Site 2 orientation (C-N linkage) (dimer peptideS453); moderate levels of agonist activity in the Site 1-Site 2orientation (N-N or C-C linkage) (dimer peptides S454 and S456); andhigh levels of agonist activity in the Site 2-Site 1 orientation (C-Nlinkage) (dimer peptide S455) (FIGS. 18A-18D).

Table 7, below, shows the HIR binding affinity and FFC assay potency ofvarious synthetic peptides, including Site 1-Site 1 dimer peptides S325,S329, S332; S333, S334, S335, S336, S337, S349, S350, S351, S352, S353,S354, S361, S362, S363, S374, S375, S376, S378, S379, S380, S381, S414,S415, S416, S417, S418, S420, and S424. These synthetic dimer peptidesexhibited properties comparable to dimer peptides 521 and 535,regardless of the orientation of the monomer subunits. In particular,synthetic Site 1-Site 2 dimer peptides S425, S453, and S459 exhibitedantagonist properties comparable to those of the Site 1-Site 2 dimerpeptides 537 and 538. Synthetic Site 1-Site 2 dimer peptides S455, S457,and S458 exhibited agonist properties comparable to the dimer peptide539. Synthetic Site 1-Site 2 dimer peptides S436, S437, S438, S454, S456act as partial agonists in the FFC assay (i.e., the peptides exhibit amaximal response of less than 100% that of insulin), which is shown inthe table as “++” and “+++”.

Table 7 also shows properties of truncated monomer and dimer peptides,and thereby indicates which N- or C-terminal residues can be deletedwithout substantial loss of HIR binding affinity (e.g., see syntheticpeptides S386 through S392, S394 through S403, and S436 through S445).Notably, certain Site 2-Site 1 dimers show IR affinities of 2*10⁻¹¹(see, e.g., S519 and S520). These peptides are also very potent in thefat cell assay (FIGS. 31A-31B) and even more potent in the HIR kinaseassay (FIGS. 32A-32B) (kinase assay described below).

TABLE 7 Site Peptide SEQ ID NO: Formula Linkage IR Sequence HIR K_(d)FEC S105 1797 F1 − 1 FHENFYDWFVRQVAKK 3.1*10⁻⁷ ++ S106 1798 F1 − 1FHENFYDWFVRQASKK 4.2*10⁻⁷ ++ S107 1799 F1 − 1 FHENFYDWFVRAVSKK10.0*10⁻⁷  + S108 1800 F1 − 1 FHENFYDWFVAQVSKK 75*10⁻⁷ + S109 1801 F1 −1 FHENFYDWFARQVSKK 2.3*10⁻⁷ ++ S110 1802 F1 − 1 FHEAFYDWFVRQVSKK2.2*10⁻⁷ ++ S111 1803 F1 − 1 FHANFYDWFVRQVSKK 33*10⁻⁷ 0 S112 1804 F1 − 1FAENFYDWFVRQVSKK 6.1*10⁻⁷ + S113 1805 F1 − 1 AHENFYDWFVRQVSKK 59*10⁻⁷ +S114 1556 F1 − fhenfydwfvrqvskk 8.3*10⁻⁶ 0 S115 1806 F1 − 1EFHENFYDWFVRQVSEE 6.5*10⁻⁷ + S116 1807 F1 − 1 FHENFYGWFVRQVSKK 1.4*10⁻⁶++ S117 1808 F2 − 1 HETFYSMIRSLAK 2.7*10⁻⁶ 0 S118 1809 F2 − 1SDGEYNAIELLS 2.4*10⁻⁶ + S119 1810 F2 − 1 SLNFYDALQLLAKK 1.8*10⁻⁶ 0 S1201811 F2 − 1 HDPFYSMMKSLLK 2.0*10⁻⁶ 0 S121 1812 F2 − 1 NSFYEALRMLSSK3.1*10⁻⁶ 0 S122 1813 F7 − HPTSKEIYAKLLK 93*10⁻⁶ 0 S123 1814 F7 −HPSTNQMLMKLFK 1.6*10⁻⁵ 0 S124 1815 F7 − HPPLSELKLFLIKK 2.3*10⁻⁵ 0 S1271816 F2 1 WSDFYSYFQGLD 1.2*10⁻⁶ 0 S128 1817 and 1818 F1-F1 C-C 1-1(FHENFYDWFVRQVSKK)₂-Dap 1.1*10⁻⁶ ++ S129 1819 F2 − 1 SSNFYQALMLLS2.9*10⁻⁶ 0 S131 1820 F1 − 1 FHENFYDWFVRQVSKK-Lig 1.2*10⁻⁶ + S137 1821 F1− 1 HENFYGWFVRQVSKK 77*10⁻⁷ 0 S145 1822 and 1823 F1-F1 C-C 1-1(FHENFYDWFVRQVSKK)₂-Lys 1.5*10⁻⁶ ++ S158 1780 F1 − 1 FHENFYDWFVRQVSK8.1*10⁻⁷ + S165 1554 F1 − 1 FYDWF  >2*10⁻⁵ 0 S166 1824 F1 − 1 FYDWFKK >2*10⁻⁵ 0 S167 1825 F1 − 1 AFYDWFAKK  >2*10⁻⁵ 31 S168 1826 F1 − 1AAAAFYDWFMAAAKK 3.8*10⁻⁶ 0 S169 1827 and 1828 F1-F1 N-N 1-112-(Lig-FHENFYDWFVRQVSKK)₂ 5.8*10⁻⁷ ++ S170 1829 and 1830 F1-F1 N-N 1-1(CGFHENFYDWFVRQVSKK)₂ 7.0*10⁻⁷ +++ (linked at cysteines) S171 1831 F1 −1 CGFHENFYDWFVRQVSKK 2.9*10⁻⁶ +++ S172 1832 and 1833 F1-F1 N-N 1-114-(Lig-FHENFYDWFVRQVSKK)₂ 4.8*10⁻⁶ +++ S173 1834 F3 − 1LDALDRLMRYFEERPSL 1.2*10⁻⁶ 0 S174 1835 F3 − 1 PLAELWAYFEHSEQGRSSAH1.6*10⁻⁵ 0 S175 1560 F1 − 1 GRVDWLQRNANFYDWFVAELG 2.3*10⁻⁷ +++ S176 1836F1 − 1 NGVERAGTGDNFYDWFVAQLH 4.7*10⁻⁷ + S177 1837 F2 − 1EHWNTVDPFYFTLFEWLRESG 2.7*10⁻⁶ 0 S178 1838 F2 − 1 EHWNTVDPFYQYFSELLRESG1.3*10⁻⁷ ++ S179 1839 F1 − 1 QSDSGTVHDRFYGWFRDTWAS 5.4*10⁻⁷ + S180 1840F1 − 1 AFYDWFAK  >2*10⁻⁵ 0 S181 1841 F1 − 1 AFYDWFA  >2*10⁻⁵ 0 S182 1842F1 − 1 AFYDWF  >2*10⁻⁵ 0 S183 1843 F1 − 1 FYDWFA  >2*10⁻⁵ 0 S184 1844 F1− 1 Ac-FYDWF  >2*10⁻⁵ 0 S214 1845 F1 − 1 AFYEWFAKK  >2*10⁻⁵ 0 S215 1846F1 − 1 AFYGWFAKK  >2*10⁻⁵ 0 S216 1847 F1 − 1 AFYKWFAKK  >2*10⁻⁵ 0 S2171848 and 1849 F2-F2 C-C 1-1 (SDGFYNAIELLS-Lig)₂-14 39*10⁻⁸ ++ S218 1850and 1851 F1-F1 C-C 1-1 (AFYDWFAKK-Lig)₂-14 1.1*10⁻⁵ 0 S219 1852 F1 − 1FHENAYDWFVRQVSKK  >2*10⁻⁵ 0 S220 1853 F1 − 1 FHENFADWFVRQVSKK  >2*10⁻⁵ 0S221 1854 F1 − 1 FHENFYAWFVRQVSKK 1.1*10⁻⁶ + S222 1855 F1 − 1FHENFYDAFVRQVSKK  >2*10⁻⁵ 0 S223 1856 F1 − 1 FHENFYDWAVRQVSKK  >2*10⁻⁵ 0S226 1857 F6 − 2 QLEEEWAGVQCEVYGRECPS 1.6*10⁻⁶ S227 1858 F1 − 1CGGFHENFYDWFVRQVSKK 5.1*10⁻⁷ ++ S228 1859 and 1860 F1-F1 N-N 1-1(CGGFHENFYDWFVRQVSKK)₂ 3.6*10⁻⁷ ++ (linked at cysteines) S229 1861 and1862 F2-F4 C-C 1-2            SDGFYNAIELLS-Lig 4.4*10⁻⁹ 0                          12 KHLCVLEELFWGASLFGYCSGKK-Lig S231 1863 and1864 F1-F1 C-C 1-1 (FHENFYDWFVRQVSKKGGG-Lig)2-14 2.7*10⁻⁷ + S232 1865and 1866 F1-F1 N-N 1-1 14-(Lig-GGGFHENFYDWFVRQVSKK)2 3.8*10⁻⁷ +++ S2331867 and 1868 F1-F2 C-C 1-1 FHENFYDWFVRQVSKK-Lig 2.6*10⁻⁷ +                  14     SDGFYNAIELLS-Lig S234 1869 F1 − 1RVDWLQRNANFYDWFVAELG 1.3*10⁻⁷ ++ S235 1870 F1 − 1 VDWLQRNANFYDWFVAELG5.3*10⁻⁸ ++ S236 1871 F1 − 1 DWLQRNANFYDWFVAELG 1.0*10⁻⁷ ++ S237 1872 F1− 1 WLQRNANFYDWFVAELG 8.5*10⁻⁷ 0 S238 1873 F1 − 1 LQRNANFYDWFVAELG8.5*10⁻⁷ 0 S239 1874 F1 − 1 QRNANFYDWFVAELG 1.3*10⁻⁶ 0 S240 1875 F1 − 1RNANFYDWFVAELG 1.4*10⁻⁶ S241 1876 F1 − 1 NANFYDWFVAELG 1.6*10⁻⁶ S2421877 F1 − 1 ANFYDWFVAELG 2.0*10⁻⁶ S243 1878 F1 − 1 NFYDWFVAELG 2.0*10⁻⁶S244 1879 F1 − 1 GRVDWLQRNANFYDWFVAELG-Lig 2.2*10⁻⁷ ++ S245 1880 F1 − 1Lig-GRVDWLQRNANFYDWFVAELG 2.2*10⁻⁷ + S246 1881 and 1882 F8-F1 C-C 3-1  ACAWFTYWNCGGGG-Lig 5.0*10⁻⁶                   14 FHENFYDWFVRQVSKK-LigS248 1883 F1 − 1 GRVDWLQRNANFYDWFVAEL 6.3*10⁻⁸ ++ S249 1884 F1 − 1GRVDWLQRNANFYDWFVAE 7.4*10⁻⁷ 0 S250 1885 F1 − 1 GRVDWLQRNANFYDWFVA8.9*10⁻⁶ 0 S251 1886 F1 − 1 GRVDWLQRNANFYDWFV 5.6*10⁻⁶ S252 1887 and1888 F2-F2 C-C 1-1 (SDGFYNAIELLS-Lig)₂-14 44*10⁻⁷ 0 S253 1889 and 1890F1-F1 C-C 1-1 (GRVDWLQRNANFYDWFVAELG-Lig)₂-14 2.2*10⁻⁸ ++ S255 1891 and1892 F2-F2 C-C 1-1 (SDGFYNAIELLSGGG-Lig)₂-14 1.6*10⁻⁶ 0 S256 1893 F6 − 2Acy-CLEEwGASL-Tic-QCSG 9.0*10⁻⁶ − S257 1894 F2 − 1 RWFNFYGYFESLLTHFS1.4*10⁻⁵ 0 S259 1895 F2 − 1 EGWDFYSYFSGLLASVT 7.7*10⁻⁶ 0 S260 1896 F2 −1 LDRQFYRYFQDLLVGFM 2.3*10⁻⁶ 0 S261 1897 F2 − 1 WGRSFYRYFETLLAQGI >2*10⁻⁵ 0 S262 1898 F4 − 1 PLCFLQELFGGASLGGYCSG 1.9*10⁻⁵ 0 S263 1899 F6− 2 WLEQERAWIWCEIQGSGCRA  >2*10⁻⁵ 0 S264 1900 F1 − 1IQGWEPFYGWFDDVVAQMFEE 1.9*10⁻⁷ 0 S265 1901 F1 − 1 TGHRLGLDEQFYWWFRDALSG1.1*10⁻⁷ 0 S266 1902 F6 − 2 Abu-CLEEwGASL-Tic-QCSG  >2*10⁻⁵ 0 S268 1903F1 − 1 RD-Hyp-FYDWEDDi 4.5*10⁻⁷ 0 S273 1904 F1-F2 C-N 1-1FHENFYDWFVRQVSKK-Lig-14-Lig-SDGFYNAIE 1.5*10⁻⁶ + LLS S278 1905F1-derived − 1 GFREGQRWYWFVAQVT  >2*10⁻⁵ 0 S281 1906 F5 −DLRVLCELFGGAYVLGYCSE 1.1*10⁻⁵ 0 S282 1907 F4-derived −HLSVGEELSWWVALLGQWAR  >2*10⁻⁵ 0 S283 1908 F4-derived −APVSTEELRWGALLFGQWAG  >2*10⁻⁵ 0 S284 1909 F6-derived −ALEEEWAWVQVRSIRSGLPL  >2*10⁻⁵ 0 S285 1910 F6-derived −WLEHEWAQIQCELYGRGCTY 8.3*10⁻⁷ S287 1911 F1 − 1 QAPSNFYDWFVREWDEE5.9*10⁻⁶ 0 S288 1912 F2 − 1 QSFYDYIEELLGGEWKK 4.3*10⁻⁶ 0 S289 1913 F2 −1 DPFYQGLWEWLRESGEE  >2*10⁻⁵ 0 S290 1914 and 1915 F1-F1 N-N 1-1 7(Lig-GGGFHENFYDWFVRQVSKK)₂ 9.0*10⁻⁷ ++ S291 1916 and 1917 F1-F1 N-N 1-19-( Lig-GGGFHENFYDWFVRQVSKK)₂ 1.2*10⁻⁶ ++ S292 1918 and 1919 F1-F1 N-N1-1 12-(Lig-GGGFHENFYDWFVRQVSKK)₂ 75*10⁻⁷ ++ S293 1920 and 1921 F1-F1N-N 1-1 13-(Lig-GGGFHENFYDWFVRQVSKK)₂ 1.2*10⁻⁷ ++ S294 1922 F1 − 1DWLQRNANFYDWFVAEL-Lig 1.3*10⁻⁷ ++ S295 1923 F1 − 1 Lig-DWLQRNANFYDWFVAEL4.8*10⁻⁷ + S300 1924 and 1925 F1-F1 C-C 1-1 (DWLQRNANFYDWFVAEL-Lig′)₂-145.0*10⁻⁸ +++ S301 1926 and 1927 F1-F1 N-N 1-114-(Lig′-DWLQRNANFYDWFVAEL)₂ 6.4*10⁻⁷ + S302 1928 F2 − 1SDGFYNA-Acy-ELLSG 8.6*10⁻⁷ 0 S303 1929 F2 − 1 SGPFYEE-Acy-ELLW-Aib-G5.7*10⁻⁶ 0 S304 1930 F2 − 1 GGSFYDD-Acy-E-Aib-LW-Aib-G 2.1*10⁻⁵ 0 S3051931 F2 − 1 N-Aib-PFYDE-Acy-DE-Cha-W-Aib-G 8.4*10⁻⁷ 0 S306 1932 F1 − 1GRVDWLQRNANFYDWFVAE-Acy-G 2.2*10⁻⁶ +++ S312 1933 and 1934 F1-F1 N-N 1-123-(Lig′-GGGFHENFYDWFVRQVSKK)₂ 2.9*10⁻⁶ ++ S313 1935 and 1936 F2-F2 C-C1-1 (SDGFYNAIELLS-Lig′)₂-23 2.4*10⁻⁷ S315 1937 F1 − 1 WFYDWFWE 6.8*10⁻⁶0 S316 1938 F10 − 1 WQGYAWLS 7.0*10⁻⁶ 0 S317 1939 F10 − 1 WFGYAWLS >2*10⁻⁵ 0 S319 1940 F1 − 1 D-Aic-D-Aib-EFYDWFDEiPq 8.7*10⁻⁷ 0 S320 1941F1 − 1 KNNKEFYEWFDEiGq 2.8*10⁻⁶ 0 S321 1942 F1 − 1 YeRD-Hyp-FYDWFDEiGg1.4*10⁻⁶ 0 S322 1943 F1 − 1 EWRD-Hyp-FYDWFDEi-Hyp-e 7.2*10⁻⁷ 0 S325 1944and 1945 F1-F1 N-N 1-1 9-(Lig′-GSLDESFYDWFERQLGKK)₂ 4.6*10⁻⁸ +++++ S3261600 F1 − ⁻¹ GIISQSCPESFYDWFAGQVSDPWWCW 59*107 − S327 1946 F2 − 1TFYSCLASLLTGTPQPNRGPWERCRKK 2.l*10⁻⁶ − S329 1947 and 1948 F1-F1 N-N 1-117-(Lig′-FHENFYDWFVRQVSKK)₂ 2.7*10⁻⁶ ++ S331 1949 F4 − 2KHLCVLEELFWGASLFGYCSGKK 1.6*10⁻⁶ 0 S332 1950 and 1951 F1-F1 C-C 1-1(GSLDESFYDWFERQLGKK-Lig′)₂-9 2.1*10⁻⁸ +++++ S333 1952 and 1953 F1-F1 N-N1-1 22-(Lig′-GSLDESFYDWFERQLGKK)₂ 1.4*10⁻⁷ ++++ S334 1954 and 1955 F1-F1N-N 1-1 22-(Lig′-GGGFHENFYDWFVRQVSKK)₂ 1.6*10⁻⁶ +++ S335 1956 and 1957F1-F1 C-C 1-1 (GSLDESFYDWFERQLGKK-Lig′)₂-22 9.8*10⁻⁸ ++++ S336 1958 and1959 F1-F1 N-N 1-1 23-(Lig′-GSLDESFYDWFERQLGKK)₂ 1.5*10⁻⁸ +++ S337 1960and 1961 F1-F1 C-C 1-1 (GSLDESFYDWFERQLGKK-Lig′)₂₋₂₃ 1.1*10⁻⁸ +++++ S3421962 F1 − 1 DLWFNAKEDMNFYDWFVWQLR 1.8*10⁻⁶ 0 S344 1963 F2 − 1EHWNTVDPFYHWISELLRESGA 2.0*10⁻⁷ 0 S345 1964 F2 − 1EHWNTVDPFYQYFAELLRESGA 2.9*10⁻⁶ 0 S349 1965 and 1966 F1-F1 N-N 1-123-(Lig′-GGGFHENFYDWFVRQVSKK)₂ 1.3*10⁻⁷ ++++ S350 1967 and 1968 F1-F1C-C 1-1 (GSLDESFYDWFERQLGKK-Lig′)₂-21 4.7*10⁻⁷ ++++ S351 1969 and 1970F1-F1 N-N 1-1 21-(Lig′-GSLDESFYDWFERQLGKK)₂ 1.4*10⁻⁶ +++ S352 1971 and1972 F1-F1 N-N 1-1 21-(Lig′-GGGFHENFYDWFVRQVSKK)₂ 6.6*10⁻⁷ +++ S353 1973and 1974 F1-F1 C-C 1-1 (GSLDESFYDWFERQLGKK-Lig′)₂-14 1.1*10⁻⁸ ++++++S354 1975 and 1976 F1-F1 N-N 1-1 14-(Lig′-GSLDESFYDWFERQLGKK)₂ 3.9*10⁻⁸++++ S359 1977 and 1978 F1-F1 N-N 1-1 9-(Lig′-DWLQRNANFYDWFVAEL)₂7.0*10⁻⁷ + S360 1979 and 1980 F1-F1 N-N 1-1 23-(Lig′-DWLQRNANFYDWFVAEL)₂9.9*10⁻⁷ S361 1981 and 1982 F1-F1 C-C 1-1 (GSLDESFYDWFERQLGKK-Lig′)₂-242.2*10⁻⁶ +++ S362 1983 and 1984 F1-F1 N-N 1-124-(Lig′-GSLDESFYDWFERQLGKK)₂ 1.1*10⁻⁷ ++++ S363 1985 and 1986 F1-F1 N-N1-1 24-(Lig′-GGGFHENFYDWFVRQVSKK)₂ 2.2*10⁻⁷ +++ S365 1987 F1 − 1RMYFSTGAPQNFYDWFVQEWD 1.0*10⁻⁵ 0 S366 1988 F1 − 1 PLRESRNFYDWFVQQLE37*10⁻⁷ 0 S368 1989 F2 − 1 RGIRSDPFYHKLSELLQGH  >2*10⁻⁵ 0 S371 1558 F1 −1 GSLDESFYDWFERQLGKK 6.3.*10⁻⁷ + S372 1990 F1 − 1 SGSLDESFYDWFERQLGKK2.0*10⁻⁷ ++ S373 1991 F1 − 1 GSLDESFYDWFERQLGKKK(S) 1.2*10⁻⁷ +++ S3741992 and 1993 F1-F1 N-N 1-1 17-(Ald-GlLDESFYDWFERQLGKK)₂ 1.8*10⁻⁷ ++++S375 1994 F1-F1 C-N 1-1 (GSLDESFYDWFERQLGKKK-Ald)-14-(Ald-GSL 2.0*10⁻++++ DESFYDWFERQLGKK) S376 1995 and 1996 F1-F1 N-N 1-119-(Ald-GSLDESFYDWFERQLGKK)₂ 1.6*10⁻⁷ ++++ S378 1997 and 1998 F1-F1 C-C1-1 (GSLDESFYDWFERQLGKKK-Ald)₂₋₁₇ 6.5*10⁻⁸ +++++ S379 1999 and 2000F1-F1 C-C 1-1 (GSLDESFYDWFERQLGKKK-Ald)₂₋₁₉ 5.6*10⁻⁸ +++++ S380 2001 and2002 F1-F1 C-C 1-1 (EEDWLQRNANFYDWFVAEL-Lig′)₂₋₉ 5.1*10⁻⁷ ++ S381 2003and 2004 F1-F1 C-C 1-1 (EEDWLQRNANFYDWFVAEL-Lig′)₂₋₂₃ 1.2*10⁻⁷ ++++ S3861559 F1 − 1 GSLDESFYDWFERQLG 3.2*10⁻⁷ + S387 2005 F1 − 1 SLDESFYDWFERQLG6.3*10⁻⁷ + S388 2006 F1 − 1 LDESFYDWFERQLG 3.4*10⁻⁷ + S389 2007 F1 − 1DESFYDWFERQLG 1.1*10⁻⁶ + S390 1794 F1 − 1 ESFYDWFERQLG 6.2*10⁻⁷ + S3912008 F1 − 1 SFYDWFERQLG 1.5*10⁻⁶ + S392 2009 F1 − 1 FYDWFERQLG 3.8*10⁻⁶0 S394 1788 F1 − 1 GSLDESFYDWFERQ 9.1*10⁻⁸ + S395 1787 F1 − 1GSLDESFYDWFERQL 8.1*10⁻⁸ ++ S396 1789 F1 − 1 GSLDESFYDWFER  >2*10⁻⁵ 0S397 2010 F1 − 1 GSLDESFYDWFE  >2*10⁻⁵ 0 S398 2011 F1 − 1 GSLDESFYDWF >2*10⁻⁵ 0 S399 1790 F1 − 1 ESFYDWFERQL 9.5*10⁻⁸ ++ S400 1791 F1 − 1ESFYDWFERQ 6.3*10⁻⁷ 0 S401 2012 F1 − 1 ESFYDWFER  >2*10⁻⁵ 0 S402 2013 F1− 1 ESFYDWFE  >2*10⁻⁵ 0 S403 2014 F1 − 1 ESFYDWF  >2*10⁻⁵ 0 S414 2015and 2016 F1-F1 C-C 1-1 (ESFYDWFERQLGK-Lig′)₂-14 3.8*10⁻⁷ ++++ S415 2017and 2018 F1-F1 C-C 1-1 (ESFYDWFERQLGK-Lig′)₂-23 1.0*10⁻⁷ ++++ S416 2019and 2020 F1-F1 N-N 1-1 14-(Lig′-ESFYDWFERQLG)₂ 9.3*10⁻⁷ +++ S417 2021and 2022 F1-F1 N-N 1-1 23-(Lig′-ESFYDWFERQLG)₂ 9.2*10⁻⁷ +++ S418 2023and 2024 F1-F1 C-C 1-1 (ESFYDWFERQLGK-Ald)₂₋₁₇ 1.2*10⁻⁷ ++++ S419 2025and 2026 F6-F6 N-N 2-2 14-(Lig′-EWLDQEWAWVQCEWGRGCPSEE)₂ 0 S420 2027 and2028 F1-F1 N-N 1-1 17-(Ald-ESFYDWFERQLG)₂ ++ S423 2029 and 2030 F1-F8C-C 1-3 ESFYDWFERQLG 6.2*10⁻⁸ 0            K ACAWPTYWNCG S425 2031 F1-F6C-N 1-2 GSLDESFYDWFERQLGKK-Lig′-14-Lig′-EWLDQ 2.4*10⁻⁹ −EWAWVQCEVYGRGCPSEE S429 2032 F6-F1 C-N 2-1EWLDQEWAWVQCEVYGRGCPSEE-Lig′-14-Lig′- 6.0*10⁻ GSLDESFYDWFERQLGKK S4322033 and 2034 F1-F6 C-C 1-2 ESFYDWFERQLGGGG 1.8*10⁻⁷ +                K      CEVYGRGCPS S433 2035 and 2036 F1-F6 C-C 1-2 ESFYDWFERQLGGGG1.1*10⁻⁷ +               K      WLDQEWAWVQ S436 2037 and 2038 F1-F6 C-C1-2    ESFYDWFERQLGGGG 5.2*10⁻¹⁰ +++                    KWLDQEWAWVQCEWGRGCPS S437 2039 and 2040 F1-F6 C-C 1-2     ESFYDWFERQLGGGG6.9*10⁻¹⁰ +++                    K LDQEWAWVQCEVYGRGCPS S438 2041 and2042 F1-F6 C-C 1-2    ESFYDWFERQLGGGG 3.0*10⁻⁸ ++                   KDQEWAWVQCEVYGRGCPS S439 2043 and 2044 F1-F6 C-C 1-2   ESFYDWFERQLGGGG4.6*10⁻⁸                  K QEWAWVQCEVYGRGCPS S440 2045 and 2046 F1-F6C-C 1-2  ESFYDWFERQLGGGG 9.9*10⁻⁸                 K EWAWVQCEVYGRGCPSS441 2047 and 2048 F1-F6 C-C 1-2 ESFYDWFERQLGGGG 1.2*10⁻⁷               K WAWVQCEVYGRGCPS S442 2049 and 2050 F1-F6 C-C 1-2ESFYDWFERQLGGGG 1.6*10⁻⁷                K  AWVQCEVYGRGCPS S443 2051 and2052 F1-F6 C-C 1-2 ESFYDWFERQLGGGG 1.7*10⁻⁷                K  WVQCEVYGRGCPS S444 2053 and 2054 F1-F6 C-C 1-2 ESFYDWFERQLGGGG1.9*10⁻⁷                K    VQCEVYGRGCPS S445 2055 and 2056 F1-F6 C-C1-2 ESFYDWFERQLGGGG 2.3*10⁻⁷                K     QCEVYGRGCPS S453 2057F1 -F6 C-N 1-2 GSLDESFYDWFERQLGKKK-Ald-17-Ald-KEWLDQ 5.7*10⁻EWAWVQCEVYGRGCPSEE S454 2058 and 2059 F1-F6 C-C 1-2     GSLDESFYDWFERQLGKKK-Ald 3.8*10⁻¹⁰ +++                            17EWLDQEWAWVQCEVYGRGCPSEEK-Ald S455 2060 F6-F1 C-N 2-1EWLDQEWAWVQCEVYGRGCPSEEK-Ald-18-Ald-G 1.1*10-⁻⁹ ++++ SLDESFYDWFERQLGKKS456 2061 and 2062 F1-F6 N-N 1-2       Ald-GSLDESFYDWFERQLGKK 2.4*10⁻⁹+++ 17 Ald-KEWLDQEWAWVQCEVYGRGCPSEE S457 2063 F6-F1 C-N 2-1WLDQEWAWVQCEVYGRGCPSGGSGGSGSLDESFYDWF 1.6*10⁻⁹ ++++ ERQLG S458 2064F6-F1 C-N 2-1 WLDQEWAWVQCEVYGRGCPSGGSGGSGSLDESFYDWF 3.2*10⁻⁹ ++++ ERQLGS459 2065 F1-F6 C-N 1-2 GSLDESFYDWFERQLGGGSGGSWLDQEWAWVQCEVYG 7.6*10⁻¹¹− RGCPS S467 2066 F6-F1 C-N 2-1 EWLDQEWAWVQCEVYGRGCPSEEK-Ald-16-Ald-G6.8*10⁻¹⁰ ++++ SLDESFYDWFERQLGKK S468 2067 F6-F1 C-N 2-1EWLDQEWAWVQCEVYGRGCPSEEK-Ald-19-Ald-G 4.0*10⁻ ++++ SLDESFYDWFERQLGKKS471 2068 F6-F1 C-N 2-1 LDQEWAWVQCEWGRGCPSESFYDWFERQLG 6.7*10⁻¹⁰ ++++S481 2069 F6-F1 C-N 2-1 HHHHHHKLDQEWAWVQCEVYGRGCPSESFYDWFERQL G S4822070 F6-F1 C-N 2-1 LDQEWAWVQCEWGRGCPSESFYDWFERQLG S483 2071 F6-F1 C-N2-1 LDEWAWVQCVEYGRGCPSESFYDWFERQLG 5.2*10⁻⁸ 0 S484 2072 F6-F1 C-N 2-1LDQEWAVQCEVYGRGCPSESFYDWFERQLG 8.7*10⁻⁸ 0 S485 2073 F6-F1 C-N 2-1LDQEWAWVCEVYGRGCPSESFYDWFERQLG 1.6*10⁻⁷ 0 S486 2074 F6-F1 C-N 2-1LDQEWAWVQCVYGRGCPSESFYDWFERQLG 5.7*10⁻⁸ 0 S487 2075 F6-F1 C-N 2-1LDQEWAWVQCEWGRCPSESFYDWFERQLG S488 2076 F6-F1 C-N 2-1LDQEWAWVQCEWGRGCSESFYDWFERQLG S489 2077 F6-F1 C-N 2-1LDQEWAWVQCEWGRGCPESFYDWFERQLG S490 2078 F6-F1 C-N 2-1LDQEWAWVQCEWGRGCESFYDWFERQLG S491 2079 F6-F1 C-N 2-1LDQEWAWVQCEWGRGCPSEFYDWFERQLG S492 2080 F6-F1 C-N 2-1LDQEWAWVQCEWGRGCPSESFYDWFRQLG S493 2081 F6-F1 C-N 2-1EWLDQEWAWVQCEVYGRGCPSEE-POX-Lys (biotin) S494 2082 F6-F1 C-N 2-1ADQEWAWVQCEVYGRGCPSESFYDWFERQLG 1.7*10⁻⁸ + S495 2083 F6-F1 C-N 2-1LAQEWAWVQCEWGRGCPSESFYDWFERQL S496 2084 F6-F1 C-N 2-1LDAEWAWVQCEWGRGCPSESFYDWFERQL S497 2085 F6-F1 C-N 2-1LDQAWAWVQCEWGRGCPSESFYDWFERQL 2.5*10⁻⁹ +++ S498 2086 F6-F1 C-N 2-1LDQEAAWVQCEVYGRGCPSESFYDWFERQL 5.6*10⁻⁸ + S499 2087 F6-F1 C-N 2-1LDQEWAAVQCEVYGRGCPSESFYDWFERQL 6.2*10⁻¹⁰ +++ S500 2088 F6-F1 C-N 2-1LDQEWAWAQCEWGRGCPSESFYDWFERQL S501 2089 F6-F1 C-N 2-1LDQEWAWVACEVYGRGCPSESFYDWFERQL S502 2090 F6-F1 C-N 2-1LDQEWAWVQCAWGRGCPSESFYDWFERQL 3.0*10⁻⁹ +++ S503 2091 F6-F1 C-N 2-1LDQEWAWVQCEAYGRGCPSESFYDWFERQL S504 2092 F6-F1 C-N 2-1LDQEWAWVQCEVAGRGCPSESFYDWFERQL S505 2093 F6-F1 C-N 2-1LDQEWAWVQCEWARGCPSESFYDWFERQL S506 2094 F6-F1 C-N 2-1LDQEWAWVQCEWGAGCPSESFYDWFERQL S507 2095 F6-F1 C-N 2-1LDQEWAWVQCEWGRACPSESFYDWFERQL S508 2096 F6-F1 C-N 2-1LDQEWAWVQCEWGRGCASESFYDWFERQL S509 2097 F6-F1 C-N 2-1LDQEWAWVQSEWGRGSPSESFYDWFERQL S510 2098 F6-F1 C-N 2-1SLEEEWAQVECEVYGRGCPSGGSGGSGLLDESFYHWF 6.2*10⁻ +++++ DRQLR S511 2099F6-F1 C-N 2-1 WLDQEWAWVQCEVYGRGCPSGGSGGSGRVDWLQRNAN 3.8*10⁻⁺⁺ FYDWFVAELGS512 2100 F6-F1 C-N 2-1 WLDQEWAWVQCEVYGRGCPSGGSGGSSQAGSAFYAWF 2.8*10⁻⁸++ DQVLRTV S513 2101 F6-F1 C-N 2-1 WLDQEWAWVQCEVYGRGCPSGGSGGSQSDAFYSGLWALIGLSDG S515 2102 F6 − 2 LDQEWAWVQCEVYGRGCPSPOX-Lys(Biotin) S516 2103F4-F1 C-N 2-1 H-Acy-CLEEwGASL-Tic-QCSGSESFYDWFERQL S517 2104 F6-F1 C-N2-1 SIEEEWAQIKCDVWGRGCPSESFYDWFERQL S518 2105 F6-F1 C-N 2-1RLEEEWAWVQCEWGRGCPSGSLDESFYDWFERQLG 1.6*10⁻¹⁰ +++++ S519 2106 F6-F1 C-N2-1 SLEEEWAQVECEVYGRGCPSGSLDESFYDWFERQLG 2.0*10⁻ +++++++ S520 2107 F6-F1C-N 2-1 SIEEEWAQIKCDVWGRGCPPGLLDESFYHWFDRQLR 2.0*10⁻¹¹ +++++ S521 2108F4-F1 C-N 2-1 HLCVLEELFWGASLFGYCSGGSLDESFYDWFERQL 2.7*10⁻⁸ + S522 2109F4-F1 C-N 2-1 HLCVLEELFWGASLFGYCSGGRVDWLQRNANFYDWFV AELG S523 2110F6-F10 C-N 2-1 WLDQEWAWVQCEVYGRGCPSDSDWAGYEWFEEQLD 4.3*10⁻⁹ ++ S524 2111F6-F1 C-N 2-1 HHHHHHKSLEEEWAQVECEVYGRGCPSGSLDESFYDW FERQLG 7,9,12,13,14,17,19, 20, 21, 22, 23, and 24 represent specific chemicallinkers (see Table 3); For FF0: 0 is no effect, + is agonist, − isantagonist. Peptides listed on 3 lines consist of two differentpeptides, linked N-N or C-C, either by chemical linkage or by beingsynthesi ed on the two branches of an amino acid with two amino groupssuch as, e.g., lysine. Acy = 1-amino-1-cyclohexanecarboxylic acid; Cha= cyclohexylalanine; Aib = 2-aminoisobutyric acid; Hyp = Hydroxyproline;Amino acids which are not capitalized are D-amino acids; Lig= Diaminopropionic acid with a 2-aminohydroxyacetyl group (CO-CH2-O-NH2)on the side chain amino group; Lig ′ = lysine with a2-aminohydroxyacetyl group (CO-CH2-O-NH2) on the side chain amino group;Ald = an aldehyde group obtained by periodate oxidation of a serine,either N-terminal or attached to the side chain amino group of lysine.

Results further indicated that S175-S175 dimer peptides (Site 1-Site 1)were less agonistic than S175 monomer peptides (++vs. +++). S175-S175dimer peptides having a C-N linkage were less agonistic or equallyagonistic as compared to S175-S175 dimer peptides having C-C or N-Nlinkages. F8-F8 dimer peptides, like the parent monomer, showed noagonist activity.

Example 5 Substrate Phosphorylation Assay (HIR Kinase)

WGA (wheat germ agglutinin)-purified recombinant human insulin receptorwas mixed with either insulin or peptide in varying concentrations insubstrate phosphorylation buffer (50 mM HEPES (pH 8.0), 3 mM MnCl₂, 10mM MgCl₂, 0.05% Triton X-100, 0.1% BSA, 12.5 μM ATP). A syntheticbiotinylated substrate peptide (Biotin-KSRGDYMTMQIG) was added to afinal concentration of 2 μg/ml. Following a 1 h incubation at RT, thereactions were stopped by the addition of 50 mM EDTA. The reactions weretransferred to Streptavidin coated 96-well microtiter plates (NUNC, Cat.No. 236001) and incubated for 1 h at RT. The plates were washed 3 timeswith TBS (10 mM Tris (pH 8.0), 150 mM NaCl).

Subsequently, a 2000-fold dilution of horseradish peroxidase (HRPO)conjugated phosphotyrosine antibody (Transduction Laboratories, Cat. No.E120H) in TBS was added. The plates were incubated for 30 min and washed3 times with TBS. TMB (3,3′,5,5′-tetramethylbenzidine; Kem-En-Tec,Copenhagen, Denmark) was added. One substrate from Kem-En-Tec was added.After 10⁻¹⁵ min, the reaction was stopped by the addition of 1% aceticacid. The absorbance, representing the extent of substratephosphorylation, was measured in a spectrophotometer at a wavelength of450 nM.

The results indicated that the potency of the Site 1-Site 2 dimer,peptide 539, was 0.1 to 1% of that of insulin in all assays tested(Table 8), and the dose-response curves (FIGS. 17A-17B) had a shapesimilar to that of insulin dose-response curves, suggesting aninsulin-like action mechanism. In addition, Site 1-Site 2 dimer peptides537 and 538 were also active as specific insulin receptor antagonists(Table 8; FIGS. 16A-16C). Notably, Site 2-Site 1 dimer peptide 539 wasmore active in the kinase assay than Site 1-Site 1 homodimer peptides521 and 535 (FIGS. 19A-19B), despite lower FFC potency (FIGS. 14A-14C;FIGS. 17A-17B). Similar results are shown in FIGS. 20A-B and FIGS.21A-B. This data suggested that homodimer and heterodimer peptides useddifferent mechanisms of action.

TABLE 8 HIR HIGF- FFC Kinase Mon./ SEQ ID Site K_(D) 1R K_(D) Pot. Pot.Pep. Link. Sequence NO: Form IR (nM) (nM) (nM) (nM) HI na na HIGF- na na1R 521 RP9- MADYKDDDDKGSLDESFYDWFE 2112 1-1 1-1 25 − A 3 1400 6aa-RQLGKKGGSGGSGSLDESFYDW RP9 FERQLGKKAAA(ETAG)PG 535 RP9-MADYKDDDDKGSLDESFYDWFE 2113 1-1 1-1 15 − A 2 1000 12aa-RQLGKKGGSGGSGGSGGSGSLD RP9 ESFYDWFERQLGKKMA(ETAG) PG 537 RP9-MADYKDDDDKGSLDESFYDWFE 2114 1-6 1-2 0.092 980 N 10 Inactive 6aa-RQLGKKGGSGGSWLDQEWAWVQ D8 CEVYGRGCPSAAA(ETAG)PG 538 RP9-MADYKDDDDKGSLDESFYDWFE 2115 1-6 1-2 0.080 710 N 10 Inactive 12aa-RQLGKKGGSGGSGGSGGSWLDQ D8 EWAWVQCEVYGRGCPSAAA(ET AG)PG 539 D8-MADYKDDDDKWLDQEWAWVQCE 2116 6-1 2-1 0.530 1500 A 10  110 6aa-WGRGCPSGGSGGSGSLDESFYD RP9 WFERQLGKKAAA(ETAG)PG A = agonist; N= antagonist; na = not applicable; Form. = formula; Mon. = constituentmonomers; Link. = linker; Pot. = potency; HI and HIGF-1R are controls;All with tags at both ends; All dimers are linked C-N; Linker sequencesare underlined.

Example 6 IR Autophosphorylation Assays

IR activation was assayed by detecting autophosphorylation of an insulinreceptor construct transfected into 32D cells (Wang et al., 1993,Science 261:1591-1594; clone 969). The IR transfected 32D cells wereseeded at 5×10⁶ cells/well in 96-well tissue culture plates andincubated overnight at 37° C. Samples were diluted 1:10 in thestimulation medium (PRIM1640 with 25 nM HEPES pH 7.2) plus or minusinsulin. The culture media was decanted from the cell culture plates,and the diluted samples were added to the cells. The plates wereincubated at 37° C. for 30 min. The stimulation medium was decanted fromthe plates, and cell lysis buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 0.5%Triton X-100, 1 mM AEBSF, 10 KIU/ml aprotinin, 50 μM leupeptin, and 2 mMsodium orthovanadate) was added. The cells were lysed for 30 min.

In the ELISA portion of the assay, the cell lysates were added to theBSA-blocked anti-IR unit mAb (Upstate Biotechnology, Lake Placid, N.Y.)coated ELISA plates. After a 2 h incubation, the plates were washed 6times with PBST and biotinylated anti-phosphotyrosine antibody (UpstateBiotechnology) is added. After another 2 h incubation, the plates wereagain washed 6 times. Streptavidin-Eu was then added, and the plateswere incubated for 1 h. After washing the plates again, EG&G Wallacenhancement solution (100 mM acetone-potassium hydrogen pthalate, pH3.2; 15 mM 2-naphtyltrifluoroacetate; 50 mM tri(n-octyl)-phosphineoxide; 0.1% Triton X-100) was added into each well, and the plates wereplaced onto a shaker for 20 min at RT. Fluorescence of samples in eachwell was measured at 615 nm using a VICTOR 1420 Multilabel Counter (EG&GWallac).

Alternatively, IR autophosphorylation was determined using a holoenzymephosphorylation assay. In accordance with this assay, 1 μl of purifiedinsulin receptor (isolated from a Wheat Germ Agglutinin ExpressionSystem) was incubated with 25 nM insulin, or 10 or 50 μM peptide in 50μl autophosphorylation buffer (50 mM HEPES pH. 8.0, 150 mM NaCl, 0.025%Triton-X-100, 5 mM MnCl₂, 50 μM sodium orthovanadate) containing 10 μMATP for 45 min at 22° C. The reaction was stopped by adding 50 μl of gelloading buffer containing β-mercaptoethanol (Bio-Rad Laboratories, Inc.,Hercules, Calif.). The samples were run on 4-12% SDS-polyacrylamidegels. Western Blot analysis was performed by transferring the proteinsonto nitrocellulose membrane. The membrane was blocked in PBS containing3% milk overnight. The membrane was incubated with anti-phosphotyrosine4G10 HRP labeled antibody (Upstate Biotechnology) for 2 h. Protein bandswere visualized using SuperSignal West Dura Extended Duration SubstrateChemiluminescence Detection System (Pierce Chemical Co.).

Example 7 Fluorescence-Based HIR Binding Assays

A. Time-Resolved Fluorescence Resonance Energy Transfer Assays

Time-resolved fluorescence resonance energy transfer assays (TR-FRET)were used for peptide competition studies. In one set of assays, monomerand dimer peptides were tested for the ability to compete withbiotinylated RP9 monomer peptide (b-RP9) for binding toHIR-immunoglobulin heavy chain chimera (sIR-Fc; Bass et al., 1996). Theassays were performed using a 384-well white microplate (NUNC) with afinal volume of 30 μl. Final incubation conditions were in 22 nM b-RP9,1 nM SA-APC (streptavidin-allophycocyanin), 1 nM Eu³⁺-sIR-Fc (LANCE™labeled, PE Wallac, Inc.), 0.05 M Tris-HCl (pH 8 at 25° C.), 0.138 MNaCl, 0.0027 M KCl, and 0.1% BSA (Cohn Fraction V). After 16-24 h ofincubation at RT, the fluorescence signal at 665 nm and 620 nm was readon a Victor² 1420 plate reader (PE Wallac, Inc.). Primary data werebackground corrected, normalized to buffer controls, and then expressedas percent of specific binding.

Results are shown in FIGS. 22A-22B. FIG. 21A shows b-RP9 competitiondata. For these figures, the Z′-factor was greater than 0.5(Z′=1−(3σ₊+3σ⁻)/|μ₊−μ⁻|; Zhang et al., 1999, J. Biomol. Screen.4:67-73), and the signal-to-background (S/B) ratio was ˜4-5. In FIG.22A, each data point represents the average of two replicate wells. Thelines represent the best fit to a four-parameter non-linear regressionanalysis of the data according to the following formula:y=min+(max−min)/(1+10̂((log IC₅₀-x)*Hillslope)). This was used todetermine IC₅₀ values.

In another set of assays, monomer and dimer peptides were tested for theability to compete with biotinylated-S175 (b-S175) or b-RP9 for bindingto sIR-Fc. The TR-FRET assays were performed in a 384-well whitemicroplate with a final volume of 30 μl. Final incubation conditionswere in 33 nM b-S175 or 22 nM b-RP9, 1 nM SA-APC, 1 nM Eu³⁺-sIR-Fc, 0.05M Tris-HCl (pH 8 at 25° C.), 0.138 M NaCl, 0.0027 M KCl, and 0.1% BSA.After 16-24 h of incubation at RT, the fluorescence signal at 665 nm and620 nm was read on a Victor² 1420 plate reader. Primary data werebackground corrected, normalized to buffer controls, and then expressedas % specific binding.

Results are shown in FIGS. 23A-23B. For these figures, each data pointrepresents the average of two replicate wells. The lines represent thebest fit to a four-parameter non-linear regression analysis of the data,which was used to determine IC₅₀ values. FIG. 23A shows b-S175competition data; FIG. 23B shows b-RP9 competition data.

B. Fluorescence Polarization Assays

Fluorescence polarization assays (FP) were used for peptide competitionstudies. In one set of assays monomer and dimer peptides were tested forthe ability to compete with fluorescein-RP9 (FITC-RP9) for binding tosoluble HIR ectodomain (sIR; Kristensen et al., 1998, J. Biol. Chem.273:17780-17786). The assays were performed in a 384-well blackmicroplate (NUNC) with a final volume of 30 μl. Final incubationconditions were 1 nM FITC-RP9, 10 nM sIR, 0.05 M Tris-HCl (pH 8 at 25°C.), 0.138 M NaCl, 0.0027 M KCl, 0.05% BGG (bovine gamma globulin),0.005% Tween-20. After 16-24 h of incubation at RT, the fluorescencesignal at 520 nm was read on an Analyst™ AD plate reader (LJLBioSystems, Inc.). Primary data were background corrected using 10 nMsIR without FITC-RP9 addition, normalized to buffer controls, and thenexpressed as percent of specific binding. The Z′-factor was greater than0.5 and the assay dynamic range was ˜125 mP. In FIGS. 24-27, each datapoint represents the average of two replicate wells. The lines representthe best fit to a four-parameter non-linear regression analysis of thedata, which was used to determine IC₅₀ values. The Z′-factor was greaterthan 0.5 and the assay dynamic range was ˜125 mP. Results are shown inFIGS. 24A-24B.

In another set of assays, monomer and dimer peptides were tested for theability to compete with FITC-RP9 for binding to soluble human insulinmini-receptor (mIR; Kristensen et al., 1999, J. Biol. Chem.274:37351-37356). The FP assays were performed in a 384-well blackmicroplate with a final volume of 30 μl. Final incubation conditionswere 2 nM FITC-RP9, 20 nM mIR, 0.05 M Tris-HCl (pH 8 at 25° C.), 0.138 MNaCl, 0.0027 M KCl, 0.001% BGG, 0.005% Tween-20. After 16-24 h ofincubation at RT, the fluorescence signal at 520 nm was read on anAnalyst™ AD plate reader. Primary data were background corrected using20 nM mIR without FITC-RP9 addition, normalized to buffer controls andthen expressed as percent of specific binding. Results are shown inFIGS. 25A-25B.

Monomers and dimer peptides were also tested for the ability to competewith fluorescein-insulin (FITC-Insulin) for binding to sIR. The FPassays were performed in a 384-well black microplate with a final volumeof 30 μl. Final incubation conditions were in 2 nM FITC-Insulin, 20 nMsIR, 0.05 M Tris-HCl (pH 8 at 25° C.), 0.138 M NaCl, 0.0027 M KCl, 0.05%BGG, 0.005% Tween-20. After 16-24 h of incubation at RT, thefluorescence signal at 520 nm was read on an Analyst™ AD plate reader.Primary data were background corrected using 20 nM sIR withoutFITC-Insulin addition, normalized to buffer controls and then expressedas percent of specific binding. Results are shown in FIGS. 26A-26B.

In other assays, peptide monomers and dimer peptides were tested for theability to compete with FITC-Insulin for binding to mIR. The FP assayswere performed in a 384-well black microplate with a final volume of 30μl. Final incubation conditions were 2 nM FITC-Insulin, 20 nM mIR, 0.05M Tris-HCl (pH 8 at 25° C.), 0.138 M NaCl, 0.0027 M KCl, 0.05% BGG(bovine gamma globulin), 0.005% Tween-20. After 16-24 h of incubation atRT, the fluorescence signal at 520 nm was read on an Analyst™ AD platereader. Primary data were background corrected using 20 nM mIR withoutFITC-RP9 addition, normalized to buffer controls and then expressed as %specific binding. Results are shown in FIGS. 27A-27B.

C. Summary

Table 9, below, summarizes the binding data calculated from competitionassays using the IR constructs, sIR-Fc, sIR, and mIR, in TR-FRET and FPformats. The data in Table 9 indicate that most dimer peptides (e.g.,S291 and S375 or S337), showed greater agonist activity than thecorresponding monomer peptides (e.g., H2C or RP9, respectively) in theFFC assay. It was previously demonstrated that an inequality betweenmonomer peptides and insulin was exhibited in competition assays wherethe assay reporter was a monomer peptide (i.e., RP9 or S175). Thisinequality was also demonstrated by dimer peptides as seen in Table 9.Table 9 further shows that Group 6 monomer peptides such as E8 (D120)were able to compete with FITC-RP9 or b-RP9 peptides for binding tosIR-Fc, but did not compete peptide ligands, such as FITC-RP9 forbinding to mIR. Experiments using different IR constructs therebyallowed differentiation of Site I peptides based on sequence motifs(i.e., Group 6 (Formula 10) vs. Group 1 (Formula 1; A6)).

TABLE 9 TARGET sIR-Fc sIR-Fc Label b-S175 b-RP9 FRET FRET Monomer SEQ IDIC50 IC50 or Dimer NO: Linkage Sequence (nM) Hill (nM) Hill H2C 2117FHENFYDWFVQRVSKK 410 −0.82 1626 −1.03 S291 1916 N-N (Lig-GGG-H2C)₂-9 81−0.96 250 −0.69 and 1917 RP9 1558 GSLDESFYDWFERQLGKK 6 −0.45 42 −0.69S375 1994 C-N (RP9-Lig)-14-(RP9-Lig) 7 −0.80 86 −0.67 S337 1960 C-C(RP9-Lig)₂-23 0.2 −0.36 14 −0.57 and 1961 S391 2008truncated-(-GSLDE)RP9(-KK) 59 −0.59 610 −0.56 S390 1794truncated(-GSLD)RP9(-KK) 27 −0.49 127 −0.49 S414 2015 C-C(truncated(-GSLD)RP9(-KK))₂-14 92 −0.62 164 −0.73 and 2016 S175 1560GRVDWLQRNANFYDWFVAELG 22 −0.58 64 −0.74 S380 2001 C-C(EE-short-S175-Lig)₂-9 10 −0.55 23 −0.64 and 2002 E8 (D120) 2118GGTVWPGYEWLRNA 755 −0.74 Insulin 59 −0.37 63 −0.46 TARGET sIR-Fc sIR mIRHIR Label ¹²⁵I- FITC-RP9 FITC-RP9 FITC-RP9 insulin FP FP FP RRA MonomerIC50 IC50 IC50 IC50 or Dimer (nM) Hill (nM) Hill (nM) Hill (nM) FFC H2C50 −0.27 37 −0.49 770 −0.89 700 + S291 12 −0.35 668 −0.38 1200 ++++ RP910 −0.41 0.03 −0.29 49 −0.53 44 +/0 S375 0.2 −0.22 91 −0.80 200 ++++S337 1 −0.37 0.2 −0.28 111 −0.70 11 +++++ S391 119 −0.49 284 −0.77 1500NN S390 19 −0.64 94 −0.94 620 + S414 0.2 −0.25 151 −0.69 NN NN S175 10−0.56 1 −0.36 167 −1.72 230 +++ S380 0.5 −0.29 27 −0.49 510 ++ E8 (D120)207 −0.49 >100000 2200 − Insulin >100000 −0.25 1250 — 172 −0.78 0.04+++++ FRET = Time-Resolved Fluorescence Resonance Energy Transfer Assay;FP = Fluorescence Polarization Assay; RRA = Radio-Receptor Assay; FFC =Free Fat Cell Assay; N-N = N-terminal linkage; C-C = C-terminal linkage;All are site 1 (formula 1) monomers or site 1-site 1 (formula 1-formula1)dimers;

Based on the functional studies outlined above, the following peptidedimers were designed.

SEQ ID Monom./ NO: Linkers Sequence 2119 F8-6aa-HLCVLEELFWGASLFGYCSGGGSGGSGSLDESFYDWF RP9 ERQL 2120 F8-12aa-HLCVLEELFWGASLFGYCSGGGSGGSGGSGGSGSLDE RP9 SFYDWFERQL 2121 D8-6aa-WLDQEWAWVQCEVYGRGCPSGGSGGSGRVDWLQRNAN S175 FYDWFVAELG 2122 D8-12aa-WLDQEWAWVQCEVYGRGCPSGGSGGSGGSGGSGRVDW 5175 LQRNANFYDWFVAELG 2123 F8-6aa-HLCVLEELFWGASLFGYCSGGGSGGSGRVDWLQRNAN 5175 FYDWFVAELG 2124 F8-12aa-HLCVLEELFWGASLFGYCSGGGSGGSGGSGGSGRVDW 5175 LQRNANFYDWFVAELG 2125 D8-6aa-HLCVLEELFWGASLFGYCSGGGSGGSSQAGSAFYAWF RP 15 DQVLRTV 2126 D8-6aa-HLCVLEELFWGASLFGYCSGGGSGGSTFYSCLASLLT RP6 GTPQPNRGPWERCR 2127 D8-6aa-HLCVLEELFWGASLFGYCSGGGSGGSQSDAFYSGLWA RP17 LIGLSDG 2128 D8-6aa-HLCVLEELFWGASLFGYCSGGGSGGSDSDWAGYEWFE Grp6 EQLDLinker sequences are underlined and in bold; Monomer sequences are shownbelow; All dimers are linked C-N.

SEQ For- ID NO: Monomer mula Site Sequence 1576 F8 4 2HLCVLEELFWGASLFGYCSG 1558 RP9 1 1 GSLDESFYDWFERQL 2129 D8 6 2WLDQEWAWVQCEVYGRGCPS 1560 S175 1 1 GRVDWLQRNANFYDWFVAELG 2130 RP15 1 1SQAGSAFYAWFDQVLRTV 1635 Rp6 2 1 TFYSCLASLLTGTPQPNRGPWERCR 2131 RP17 1 1QSDAFYSGLWALIGLSDG 1595 Group 6 10 1 DSDWAGYEWFEEQLD

Example 8 Peptide Fusions to the Maltose Binding Protein

A. Cloning

The transfer of interesting peptide sequences from phage display tomaltose binding protein (MBP) fusions is desirable for several reasons.First, to obtain a more sensitive affinity estimate, the polyvalency ofphage display peptides should be converted to a monovalent system. Forthis purpose, the peptide sequences are fused to MBP that generallyexists as a monomer with no cysteine residues. Second, competitionexperiments can be carried out with the same or different peptides, onephage displayed and the other fused to MBP. Lastly, purified peptidescan be obtained by cleavage of the fusion protein at a site engineeredin the DNA sequence.

FIG. 28 shows a schematic drawing of the MBP-peptide construct. In theconstruct, the N-terminus of the peptide sequence is fused to theC-terminus of the MBP. Two peptide-flanking epitope tags are included, ashortened-FLAG® at the N-terminus and E-Tag at the C-terminus. Thecorresponding gene fusion was generated by ligating a vector fragmentencoding the MBP in frame with a PCR product encoding the peptide ofinterest. The vector fragment was obtained by digesting the plasmidpMAL-c2 (New England Biolabs) with EcoRI and HindIII and then treatingthe fragment with shrimp alkaline phosphatase (SAP; Amersham). Thedigested DNA fragment was resolved on a 1% agarose gel, excised, andpurified by QIAEXII (QIAGEN). The 20-amino acid peptide sequences ofinterest were initially encoded in the phage display vector pCANTAB5E(Pharmacia). To obtain these sequences, primers were synthesized whichanneal to sequences encoding the shortened FLAG® or E-Tag epitopes andalso contain the required restriction enzyme sites EcoRI and HindIII.PCR products were obtained from individual phage clones and digestedwith restriction enzymes to yield the insert fragment. The vector andinsert were ligated overnight at 15° C. The ligation product waspurified using QIAquick spin columns (QIAGEN) and electroporations wereperformed at 1500 v in an electroporation cuvette (0.1 mm gap; 0.5 mlvolume) containing 10 ng of DNA and 40 μl of E. coli strain ER2508 (RR1lon:mini Tn10 (Tet^(r)) (malB) (argF-lac) U169 Pro⁺ zjc::Tn5(Kan^(r))fhuA2) electrocompetent cells (New England Biolabs). Immediately afterthe pulse, 1 ml of pre-warmed (40° C.) 2xYT medium containing 2% glucose(2xYT-G) was added and the transformants were grown at 37° C. for 1 h.Cell transformants were plated onto 2xYT-AG plates and grown overnightat 37° C. Sequencing confirmed the clones contained the correctconstructs.

B. Small-Scale Expression of Soluble MBP-Peptide Fusion Proteins

E. coli ER2508 (New England Biolabs) carrying the plasmids encodingMBP-peptide fusion proteins were grown in 2xYT-AG at 37° C. overnight(250 rpm). The following day the cultures were used to inoculate media(2x YT containing-G) to achieve an OD₆₀₀ of 0.1. When the culturesreached an OD₆₀₀ of 0.6, expression was induced by the addition of IPTGto a final concentration of 0.3 mM and then cells were grown for 3 h.The cells were pelleted by centrifugation and samples from total cellswere analyzed by SDS-PAGE electrophoresis. The production of the correctmolecular weight fusion proteins was confirmed by Western blot analysisusing the monoclonal antibody anti-E-Tag-HRP conjugate (Pharmacia).

C. Large-Scale Expression of Soluble MBP-Peptide Fusion Proteins

E. coli ER2508 carrying plasmids encoding the MBP-peptide fusionproteins were grown in 2xYT-AG media for 8 h (250 rpm, 37° C.). Thecultures were subcultured in 2xYT-AG to achieve an OD₆₀₀ of 0.1 andgrown at 30° C. overnight. This culture was used to inoculate afermentor with medium of following composition (g/l): glucose (3.00);(NH₄)₂SO₄ 5.00; MgSO₄.7H₂O (0.25); KH₂PO₄ (3.00); citric acid (3.00);peptone (10.00); and yeast extract (5.00); pH 6.8.

The culture was grown at 700 rpm, 37° C. until the glucose from themedium was consumed (OD₆₀₀=˜6.0-7.0). Expression of the fusion proteinwas induced by the addition of 0.3 mM IPTG and the culture was grown for2 h in fed-batch mode fermentation with feeding by 50% glucose at aconstant rate of 2 g/l/h. The cells were removed from the medium bycentrifugation. Samples of the cell pellet were analyzed by SDS-PAGEfollowed by the Western blot analysis using the mouse monoclonalantibody anti-E-Tag-HRP conjugate (Pharmacia) to visualize the expressedproduct.

D. Purification

The cell pellets were disrupted mechanically by sonication or chemicallyby treatment with the mild detergent Triton X-100. After removal of celldebris by centrifugation, the soluble proteins were prepared forchromatographic purification by dilution or dialysis into theappropriate starting buffer. The MBP fusions were initially purifiedeither by amylose affinity chromatography or by anion exchangechromatography. Final purification was performed using anti-E-Tagantibody affinity columns (Pharmacia). The affinity resin wasequilibrated in TBS (0.025 M Tris-buffered saline, pH 7.4) and the boundprotein was eluted with Elution buffer (100 mM glycine, pH 3.0). Thepurified proteins were analyzed for purity and integrity by SDS-PAGE andWestern blot analysis according to standard protocols.

For MBP fusions, IR agonist activity was observed for the Site 1-Site 1dimer peptides shown in Table 10, below. Additional binding data for theMBP fusions are shown in Table 11, also below.

TABLE 10 Monomer/ SEQ ID Site Fus. MW Fus. Linker Sequence NO: Form.Act. IR Conc. (kDa) K_(d)(HIR) 426 D8 MBP . . . NNNNLGIEGRISEFIEGRAQPAMA2132 6 N 2 0.76 52.2 1.4 × 10⁻⁶ WLDQEWAWVQCEVYGRGCPSMA(ETAG)AA 429D8-6aa-D8 MBP. . . NNNNLGIEGRISEFIEGRAQPAMAW 2133 6-6 N-N 2-2 3.2 55.31.3 × 10⁻⁶ LDQEWAWVQCEVYGRGCPSGGSGGSKWLDQEWAW VQCEVYGRGCPSMA(ETAG)AA 430H20-4aa-RB6 MBP . . . NNNNLGIEGRISEFIEGRDYKDDD 2134 1-6 A- 1-1 0.17 54.52.1 × 10⁻⁶ DKFHENFYDWFVRQVSGSGSLDALDRLMRYFEER PSLETAG 431 H20-6aa-F8MBP. . . NNNNLGIEGRISEFIEGRDYKDDDK 2135 1-4 A-N 1-2 3.3 54.8 4.7 × 10⁻FHENFYDWFVRQVSGGSGGSHLCVLEELFWGASL FGYCSGAAA(ETAG)AA 432 H20-12aa-F8MBP-NNNNLGIEGRISEFIEGRDYKDDDKFHENF 2136 1-4 A-N 1-2 2.9 55.5 3.5 × 10⁻YDWFVRQVSGGSGGSGGSGGSHLCVLEELFWGAS LFGYCSGAAA(ETAG)AA 433 H20-9aa-F8 MBP. . . NNNNLGIEGRISEFIEGRDYKDDD 2137 1-4 A-N 1-2 2.8 55.2 2.1 × 10⁻⁸KFHENFYDWFVRQVSGGSGGSGGSHLCVLEELFW GASLFGYCSGMA(ETAG)AA 434 G3-12aa-G3MBP . . . NNNNLGIEGRISEFIEVRAQPAMA 2138 1-1 N-N 1-1 0.01 56 3.2 × 10⁻⁶RGGGTFYEWFESALRKHGAGGGSGGSGGSGGSRG GGTFYEWFESALRKHGAGAAA(ETAG)AA 436H2C-9aa-H2C MBP . . . NNNNLGIEGRISEFIEGRAQPAMA 2139 1-1 A 1-1 1.1 54.24.1 × 10⁻ FHENFYDWFVRQVSGGSGGSGGSFHENFYDWFVR QVSMA(ETAG)AA 437 H2C MBP .. . NNNNLGIEGRISEFIEGRAQPAMA 2140 1 N-N 1 0.3 51.5 8.3 × 10⁻⁶FHENFYDWFVRQVSAAA(ETAG)AA 427 G3-6aa-G3 MBP . . .NNNNLGIEGRISEFIEGRAQPAMA 2141 1-1 N-N 1-1 0.02 55.3 3.3 × 10⁻RGGGTFYEWFESTLRKHGAGGGSGGSRGGGTFYE WFESALRKHGAGAAA(ETAG)AA 435H2C-3aa-H2C- MBP . . . NNNNLGIEGRISEFIEGRAQPAMA 2142 1-1-1 A-A-A 1-1-12.1 55.5 2.0 × 10⁻⁶ 3aa-H2C FHENFYDWFVRQVSGGSFHENFYDWFVRQVSGGSFHENFYDWFVRQVSAAA(ETAG)AA 439 H2C-6aa-H2C MBP . . .NNNNLGIEGRISEFIEGRAQPAMA 2143 1-1 A-A 1-1 1.4 53.9 5.5 × 10⁻⁷FHENFYDWFVRQVSGGSGGSFHENFYDWFVRQVS (ETAG)AA 449 H2C-12aa-H2C MBP . . .NNNNLGIEGRISEFIEGRAQPAMA 2144 1-1 1-1 1.5 51.8 6.2× 10⁻⁷FHENFYDWFVRQVSGGSGGSGGSGGSAQPAMAFH ENFYDWFVRQVSAAA(ETAG)AA 452 G3 MBP .. . NNNNLGIEGRISEFIEGRAQPAMA 2145 1 1 0.15 48.8 7.8 × 10⁻⁷RGGGTFYEWFESALRKHGAGAAA(ETAG)AA 463 H2C-3aa-H2C MBP . . .NNNNLGIEGRISEFIEGRAQPAMA 2146 1-1 A-A 1-1 1.8 50.1 9.6 × 10⁻⁷FHENFYDWFVRQVSGGSFHENFYDWFVRQVSMA (ETAG)AA 464 LF-H2C MBP . . .NNNNLGIEGRISEFIEGRDYKDDD 2147 1 1 0.045 48.4 3.9 × 10⁻⁸DKFHENFYDWFVRQVSAA(ETAG)AA 446 LF-F8 MBP . . . NNNNLGIEGRISEFIEGRDYKDDD2148 1 2 1.9 49.1 7.7 × 10⁻⁷ DKHLCVLEELFWGASLFGYCSGAAA(ETAG)AA 459SF-RB6 MBP . . . NNNNLGIEGRISEFGSADYKDLDA 2149 3 1 0.069 48.1 7.7 × 10⁻⁸LDRLMRYFEERPSLAAA(ETAG)AA MBP* IacZ ** na 5.1 50  >1 × 10⁻⁵ *MBP(negative control for the fusions) is fused to a small fragment ofbeta-galactosidase (lacZ); **MBP-lacZ fusion protein was derived fromthe plasmid pMal-c2 as purchased form NEB. Fus. = fusion; Act.= activity; Conc. = concentration; N = Antagonist; A = Agonist; LF= Long FLAG  ® epitope (DYKODDOK; SEQ ID NO:1777); SF = Short FLAG ® epitope (DYKD; SEQ ID NO:1545); na = not applicable; Form. = formula;All dimers are linked C-N; Linker sequences are underlined.

TABLE 11 High SEQ conc. Kd Fu- Monomer/ ID Site tested (HIR) sion LinkerSequence NO: Form. IR (μM) μM 431− H2C-6aa-F8 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDKFHENFYDWFVRQVSGGSGGSHL 2150 1-6 1-2 0.2 0.033CVLEELFWGASLFGYCSGAAA(ETAG)AA 431+ H2C-6aa-F8DYKDDDKFHENFYDWFVRQVSGGSGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)A 2151 1-6 1-20.2 0.0074 A 432− H2C-12aa-F8 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDKFHENFYDWFVRQVSGGSGGSGG 2152 1-6 1-2 0.2 0.02SGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)AA 432+ H2C-12aa-F8DYKDDDKFHENFYDWFVRQVSGGSGGSGGSGGSHLCVLEELFWGASLFGYCSGAAA 2153 1-6 1-20.2 0.0038 (ETAG)AA 433− H2C-9aa-F8 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDKFHENFYDWFVRQVSGGSGGSGG 2154 1-6 1-2 0.2 0.03SHLCVLEELFWGASLFGYCSGAAA(ETAG)AA 433+ H2C-9aa-F8 DYKDDDKFHENFYDWFVRQVSGGSGGSGGS 2155 1-6 1-2 0.2 0.004HLCVLEELFWGASLFGYCSGAAA(ETAG)AA The concentrations of these fusions varydepending on the expression quality. There are 2 sets of each fusion:uncleaved (−) and cleaved with factor Xa (+). The fusion proteins are inTris buffer (20 mM Tris, 200 mM NaCl, 1 mM EDTA, 50 mM maltose, pH 7.5)and the cleaved fusions (+) are in the same Tris buffer (500 μl) + 12 μgFactor Xa. (Source of Factor Xa: New England Biolabs). Conc.= concentration; Form. = formula; All dimers are linked C—N; Linkersequences are underlined.

E. BIAcore Analysis

For BIAcore analysis of fusion protein and synthetic peptide binding toinsulin receptor, insulin (50 μg/ml in 10 mM sodium acetate buffer pH 5)was immobilized on the CM5 sensor chip (Flowcell-2) by amine coupling.Flowcell-1 was used for background binding to correct for anynon-specific binding. Insulin receptor (450 nM) was injected into theflow cell and the binding of IR to insulin was measured in resonanceunits (RUs). Receptor bound to insulin gave a reading of 220 RU. Thesurface was regenerated with 25 mM NaOH. Pre-incubation of receptor withinsulin in a tube at RT completely abrogated the response units to 16RU. Thus, the system was validated for competition studies. Severalmaltose-binding fusion proteins, peptides, and rVabs were pre-incubatedwith insulin receptor before injecting over the insulin chip forcompetition studies. The decrease in binding/resonance units indicatesthat several MBP-fusion proteins can block the insulin-binding site. Theresults are shown in Tables 12 and 13. The amino acid sequences referredto in the tables are identified in FIGS. 8 and 9A-9B, except the 447 and2A9 sequences, which are shown below.

TABLE 12 BIAcore Results-Fusion Proteins Compete for Binding to IRResult Incubation Mixtures (RUs) Sequence Type Controls Insulin Receptor220 Positive Control (IR) 450 nM Insulin (8.7 μM)  16 Negative ControlMBP Fus. A7 (20A4)-MBP  43 Formula 6 Motif Prots. (4.1 μM) + IR D8-MBP 56 Formula 6 Motif (1.6 μM) + IR D10-MBP  81 Formula 11 Motif (3.4 μM)+ IR 447-MBP 195 hGH Pept. Fus. (11.5 μM) + IR MBP (13 μM) + IR 209Negative Control The A7 (20A4), D8, and D10 peptide sequence are shownin FIGS. 8 and 9A-9B. The 447 peptide sequence is:LCQRLGVGWPGWLSGWCA(SEQ ID NO: 2156).

TABLE 13 BIAcore Results-Synthetic peptides compete for binding to IRIncubation % Result Mix Binding (RUs) Sequence Type IR 100 128 Positivecontrol IR + 20D1 41 51.8 Formula 1 Motif IR + D8 33 41.6 Formula 6Motif IR + 20C11 38 49 Formula 2 Motif (bkg high) IR + H2 27 34.6 IGF(phosphorylated band) IR + 2A9 100 128 IGF(bkg high) IR + 20A4 33 41.8Formula 6 Motif IR + p53wt 97 124.5 P53 wild type The concentration ofeach peptide was about 40 μM and the concentration of IR was 450 nM. The20D1, 20A4, and D8 peptide sequences are shown in FIGS. 8 and 9A-9B. Theremaining peptide sequences are as follows: 447 = LCQRLGVGWPGWLSGWCA(SEQ ID NO: 2156); 2A9 = LCQSWGVRIGWLTGLCP (SEQ ID NO: 2157); 20C11= DRAFYNGLRDLVGAVYGAWD (SEQ ID NO: 1659); H2 = VTFTSAVFHENFYDWFVRQVS(SEQ ID NO: 1784).

Regarding preparation of a Site 1 agonist comprising two D117 (H2C)peptides, a linker of only 3 amino acids (12 Å) provided a ligand ofgreater affinity for Site 1 of IR than a corresponding ligand preparedwith a 9 amino acid (36 Å) linking region (FIG. 29).

F. Stimulation of Autophosphorylation of IR by MBP-Fusion Peptides

MBP fusion peptides were prepared as described above, and then assayedfor autophosphorylation of a insulin receptor construct transfected into32D cells (Wang et al., 1993; clone 969) (see Example, above). Theresults of these experiments shown in FIG. 30 indicate that the H2Cmonomer and H2C-H2C homodimer peptides stimulate autophosphorylation ofIR in vivo. H2C dimer peptides (Site 1-Site 1) with a 6 amino acidlinker (H2C-6aa-H2C) were most active in the autophosphorylation assay.Other active dimer peptides are also shown in FIG. 30, particularlyH2C-9aa-H2C, H2C-12aa-H2C, H2C-3aa-H2C, and F8.

G. Insulin Receptor Binding Affinity and Fat Cell Potency of MBP-FusionPeptides

Results of assays to determine binding affinity for insulin receptor andfat cell potency of the MBP-fusion peptides are shown in Table 14,below.

TABLE 14 SEQ ID Site HIR Kd Peptide NO: Formula IR Sequence (mol/l) FFCRB426 2158 F6 2 MBP . . . NNNNLGIEGRISEFIEGR AQPAMA WLDQEWAWVQCEVYGRGCPSAAA(ETAG)AA  1.4*10⁻⁶ RB429 2159 F6-F6 2-2 MBP . . .NNNNLGIEGRISEFIEGRAQPAMAWLDQEWAWVQCEVYGRGCPSGGSGGSKWLDQEWAWVQCEVYGRGCPSAAA(ETAG)AA 1.3*10⁻⁶ RB505M 2160 F4 2 MBP . . . NNNNLGIEGRISEFIEGRDYKDDDDKHLCVLEELFWGASLFGYCSGAAA(ETAG)AA RB517M 2161 F4-F4 2-2 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDK HLCVLEELFWGASLFGYCSGGGSGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)AA RB515 2162 F4-F4 2-2 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDK HLCVLEELFWGASLFGYCSGGGSGGSGGSGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)AA RB510 2163 F4-F4-F4 2-2-2 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDK HLCVLEELFWGASLFGYCSGGGSGGSHLCVLEELFWGASLFGYCSGGGSGGS HLCVLEELFWGASLFGYCSGAAA(ETAG)AA RB437 2164 F11 MBP . . . NNNNLGIEGRISEFIEGRAQPAMA FHENFYDWFVRQVSAAA(ETAG)AA  8.3*10⁻⁶RB463 2165 F1-F1 1-1 MBP . . .NNNNLGIEGRISEFIEGRAQPAMAFHENFYDWFVRQVSGGSFHENFYDWFVRQVSAAA(ETAG)AA 9.6*10⁻⁷ RB439 2166 F1-F1 1-1 MBP . . . NNNNLGIEGRISEFIEGRAQPAMAFHENFYDWFVRQVSGGSGGSFHENFYDWFVRQVS-ETAG  5.5*10⁻⁷ RB436 2167 F1-F1 1-1MBP . . .NNNNLGIEGRISEFIEGRAQPAMAFHENFYDWFVRQVSGGSGGSGGSFHENFYDWFVRQVSAAA(ETAG)AA 4.1*10⁻⁷ RB449 2168 F1-F1 1-1 MBP . . . NNNNLGIEGRISEFIEGRAQPAMAFHENFYDWFVRQVSGGSGGSGGSGGS AQPAMAFHENFYDWFVRQVSAAA(ETAG)AA 6.2*10⁻⁷ RB435 2169 F1-F1-F1 1-1-1 MBP . . .NNNNLGIEGRISEFIEGRAQPAMAFHENFYDWFVRQVSGGSFHENFYDWFVRQVSGGSFHENFYDWFVRQVSAAA(ETAG)AA 2.0*10⁻⁶ RB502 2170 F1 1 MBP . . . NNNNLGIEGRISEFIEGRDYKDDDDKVRVDWLQRNANFYDWFVAELVAAA(ETAG)AA RB508M 2171 F1-F1 1-1 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDKVRVDWLQRNANFYDWFVAELGGGSGGSGRVDWLQRNANFYDWFVAELGAAA(ETAG)AA RB509M 2172 F1-F1 1-1 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDKVRVDWLQRNANFYAWFVAELGGGSGGSGGSGGSGRVDWLQRNANFYDWFVAELGAAA(ETAG)AA RB452 2173 F1 1 MBP . . .NNNNLGIEGRISEFIEGRAQPAMARGGGTFYEWFESALRKHGAGAAA(ETAG)AA  7.8*10⁻⁷ RB4272174 F1-F1 1-1 MBP . . .NNNNLGIEGRISEFIEGRAQPAMARGGGTFYEWFESTLRKHGAGGGSGGSRGGGTFYEWFESALRKHGAGAAA(ETAG)AA 3.3*10⁻⁶ RB434 2175 F1-F1 1-1 MBP . . . NNNNLGIEGRISEFIEVRAQPAMA 3.2*10⁻⁶RGGGTFYEWFESALRKHGAGGGSGGSGGSGGSRGGGTFYEWFESALRKHGAGAAA(ETAG)AA RB5132176 F1 1 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDKGSLDESFYDWFERQLGKKAA(ETAG)AA RB516 2177 F1-F11-1 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDKGSLDESFYDWFERQLGKKGGSGGSGSLDESFYDWFERQLGKKAAA(ETAG)AARB512 2178 F1-F1 1-1 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDKGSLDESFYDWFERQLGKKGGSGGSGGSGGSGSLDESFYDWFERQLGKKAAA(ETAG)AARB464 2179 F1 1 MBP . . . NNNNLGIEGRISEFIEGRDYKDDDDKFHENFYDWFVRQVSAA(ETAG)AA  3.8*10⁻¹⁸ RB446 2180 F4 2 MBP . . .NNNNLGIEGRISEFIEGRDYKDDDDKHLCVLEELFWGASLFGYCSGAAA(ETAG)AA  7.7*10⁻⁷RB459 2181 F3 1 MBP . . .NNNNLGIEGRISEFGSADYKDLDALDRLMRYFEERPSLAAA(ETAG)AA  7.7*10⁻⁸ RB430 2182F1-F3 1-1 MBP . . . NNNNLGIEGRISEFIEGRDYKDDDDKFHENFYDWFVRQVSGGSGGSLDALDRLMRYFEERPSLETAG  2.1*10⁻⁶ − RB430 2183 F1-F3 1-1 cleavedDYKDDDKFHENFYDWFVRQVSGSGSLDALDRLMRYFEERPSLAAA(ETAG)AA   ~4*10⁻⁹ RB4312184 F1-F4 1-2 MBP . . . NNNNLGIEGRISEFIEGRDYKDDDKFHENFYDWFVRQVSGGSGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)AA  4.710⁻⁸ − RB431 2185 F1-F4 1-2 cleavedDYKDDDKFHENFYDWFVRQVSGGSGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)AA   ~8*10⁻⁹RB432 2186 F1-F4 1-2MBP-NNNNLGIEGRISEFIEGRDYKDDDKFHENFYDWFVRQVSGGSGGSGGSGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)AA  3.5*10⁻⁸ − RB432 2187 F1-F4 1-2 cleavedDYKDDDKFHENFYDWFVRQVSGGSGGSGGSGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)AA  ~6*10⁻⁹ RB433 2188 F1-F4 1-2 MBP . . . NNNNLGIEGRISEFIEGRDYKDDDKFHENFYDWFVRQVSGGSGGSGGS HLCVLEELFWGASLFGYCSGAAA(ETAG)AA  2.1*10⁻⁸ RB5082189 F1-F1 1-1DYKDDDDKVRVDWLQRNANFYDWFVAELGGGSGGSGRVDWLQRNANFYDWFVAELGAAAGAPVPYPDPLEPRSA 1.5*10⁻⁷ ++ RB509 2190 F1-F1 1-1DYKDDDDKVRVDWLQRNANFYAWFVAELGGGSGGSGGSGGSGRVDWLQRNANFYDWFVAELGAAAGAPVPYPDPLEPRAA 5.5*10⁻⁸ ++ RB505 2191 F4 2 DYKDDDDKHLCVLEELFWGASLFGYCSGAAA(ETAG)AA 4.8*10⁻⁷ − RB517 2192 F4-F4 2-2DYKDDDDKHLCVLEELFWGASLFGYCSGGGSGGSHLCVLEELFWGASLFGYCSGAAA(ETAG)AA 6.0*10⁻⁶ − RB521 2193 F1-F1 1-1MADYKDDDDKGSLDESFYDWFERQLGKKGGSGGSGSLDESFYDWFERQLGKKAAA(ETAG)PG 4.4*10⁻⁸ +++++ RB535 2194 F1-F1 1-1MADYKDDDDKGSLDESFYDWFERQLGKKGGSGGSGGSGGSGSLDESFYDWFERQLGKKAAA(ETAG)PG~1.0*10⁻⁷ +++++ RB540 2195 F6 2MADYKDDDDKWLDQEWAWVQCEVYGRGCPSAAA(ETAG)PG ~1.0*10⁻⁷ RB539 2196 F6-F1 2-1MADYKDDDDKWLDQEWAWVQCEVYGRGCPSGGSGGSGSLDESFYDWFERQLGKKAAA(ETAG)PG   7*10⁻⁰ ++++ RB537 2197 F1-F6 1-2MADYKDDDDKGSLDESFYDWFERQLGKKGGSGGSWLDQEWAWVQCEVYGRGCPSAAA(ETAG)PG 5.9*10⁻¹¹ − RB538 2198 F1-F6 1-2MADYKDDDDKGSLDESFYDWFERQLGKKGGSGGSGGSGGSWLDQEWAWVQCEVYGRGCPSAAA(ETAG)PG 1.7*10⁻¹¹ − RB626 2199 F6-F1 2-1MADYKDEIEAEWGRVRCLVYGRCVGGGGSGGSGGSGGSGSLDESFYDWFERQLGKKAAA(ETAG)PG 3.0*10⁻¹⁰ +++ RB625 2200 F6-F1 2-1MADYKDDDDKWLDQEWAWVQCEVYGRGCPSQPPPPDITTHRADPQGSLDESFYDWFERQLGKKAAA(ETAG)PG 3.8*10⁻¹⁰ +++++ RB622 2201 F6-F1 2-1MADYKDDDDKWLDQEWAWVQCEVYGRGCPSTPKPPTPPPLSADGSLDESFYDWFERQLGKKAAA(ETAG)PG 1.0*10⁻⁹ ++++ RB596 2202 F1 1 MQNDDGSLDESFYDWFERQLGHHHHHHPG  9.4*10⁻⁸RB569 2203 F1 1 MGSLDESFYDWFERQLGEEEGGDHHHHHHPG  2.1*10⁻⁷ RB570 2204 F11 MQNDDGSLDESFYDWFERQLGEEEGGDHHHHHHPG  2.5*10⁻⁸ ETAG = GAPVPYPDPLEPR(SEQID NO: 2205); MBP . . . NNNNL = fusion junction to MBP at c-terminus ofMBP; All dimers are linked C—N.

Example 9 In Vivo Assays for Insulin Agonists

To test the in vivo activity of dimer peptide S519, an intravenous bloodglucose test was carried out on Wistar rats. Male Mol:Wistar rats,weighing about 300 g, were divided into two groups. A 10 μl sample ofblood was taken from the tail vein for determination of blood glucoseconcentration. The rats were anaesthetized with Hypnorm/Dormicum att=−30 min and blood glucose was measured again at t=−20 min and at t=0min. After the t=0 sample was taken, the rats were injected into thetail vein with vehicle or test substance in an isotonic aqueous bufferat a concentration corresponding to a 1 ml/kg volume of injection. Bloodglucose was measured at times 10, 20, 30, 40, 60, 80, 120, and 180 min.The Hypnorm/Dormicum administration was repeated at 20 min intervals.Results shown in FIG. 33 demonstrate that the S519 (at 20 nmol/kg)peptide lowered blood glucose levels similar to levels observed forhuman insulin (at 2.5 nmol/kg) (n=8). The S519 peptide and human insulinshowed comparable in vivo effects, both in magnitude and onset ofresponse (FIG. 33).

Example 10 IGF-1 Binding Peptides

Three major groups of peptide IGF-1-binding peptides were obtained fromIGF-1R panning experiments: Site 1 A6 (FyxWF) (SEQ ID NO:1596); Site 1B6 (FyxxLxxL) (SEQ ID NO:1732), and Site 2 (cysteine loops). See Beasleyet al. International Application PCT/US00/08528, filed Mar. 29, 2000,and Beasley et al., U.S. application Ser. No. 09/538,038, filed Mar. 29,2000. Active peptides included 20E2 and RP6 (B6-like; Formula 2), S175(A6-like; Formula 1), G33 (A6-like; Formula 1), RP9 (A6-like; Formula1), D815 (Site 2), and D8B12 (Site 2) peptides. The IGF-1 bindingpeptides were analyzed by various assays, described as follows.

A. Phage Competition

Phage competition studies were performed with Site 1 (RP9) and Site 2(D815) monomer peptides. Plates were coated with IGF-1R (100 ng/well incarbonate buffer, pH 9.6) overnight at 4° C. Wells were blocked with 4%non-fat milk in PBS for 60 min at room temperature. One hundredmicroliters of rescued phage were added to each well. Peptides invarying concentrations were added and the mixtures were incubated for 2h at room temperature. Plates were washed three times with PBS and 100μl of anti-M13 antibody conjugated to horseradish peroxidase was addedto each well. The labeled antibody was incubated at room temperature for60 min. After washing, 100 μl of ABTS was added per well and the platesread in a microtiter reader at 450 nM.

Phage included RP9 (A6-like; Formula 1); RP6 (B6-like; Formula 2); D8B12(Site 2); and D815 (Site 2). Peptides included RP9 and D815.

Site SEQ Pep- IGF- ID tide Formula 1R Sequence NO: D8B12 6 2WLEQERAWIWCEIQGSGCRA 1884 D815 6 2 WLDQERAWLWCEISGRGCLS 2206 RP6 2 1TFYSCLASLLTGTPQPNRGPWERCR 1635 RP9 1 1 GSLDESFYDWFERQLG 1559

Results shown in FIGS. 34A-34E demonstrate that that RP9 and D815peptides competed both Site 1 and Site 2 phage. These results illustratethe allosteric nature of the interaction with IGF-1R.

Phage competition studies were also performed with Site 2-Site 1 dimerpeptides containing 6- or 12-amino acid linkers. Plates were coated withIGF-1R (100 ng/well in carbonate buffer, pH 9.6) overnight at 4° C.Wells were blocked with 4% non-fat milk in PBS for 60 min at roomtemperature. One hundred microliters of rescued phage were added to eachwell. Peptides in varying concentrations were added and the mixtureincubated for 2 h at room temperature. Plates were washed three timeswith PBS and 100 μl of anti-M13 antibody conjugated to horseradishperoxidase was added to each well. The labeled antibody was incubatedfor 60 min at room temperature. After washing, 100 μl of ABTS was addedper well and the plates read in a microtiter reader at 450 nM. Phageincluded RP9, RP6, D8B12, and D815. Peptides included D815-6L-RP9 andD815-12L-RP9. Linker sequences are underlined and shown below.

Site SEQ ID Peptide Formula IGF-1R Sequence NO: D815-6L- 6-1 2-1LDQERAWLWCEISGRGCLSGGSGGSGSLDESFYDWFERQLGKK 2207 RP9 D815- 6-1 2-1WLDQERAWLWCEISGRGCLSGGSGGSGGSGGSGSLDESFYDW 2208 12L-RP9 FERQLGKK

D8B12, D815, RP6, and RP9 amino acid sequences are shown in the previoussection. Results shown in FIGS. 35A-35E demonstrate that dimers competedboth Site 1 and Site 2 phage. This indicates that both dimer units wereactive at IGF-1R.

B. IGF-1 Proliferation Assays FDC-P2 cells expressing the IL-3 and humanIGF-1R receptors were grown in RPMIk-1640 medium supplemented with 15%fetal bovine serum (FBS) and 5% WEHI conditioned medium (containingIL-3) in accordance with routine methods. Prior to an experiment, thecells were pelleted and washed two times in PBS. Following this, cellswere resuspended in RPMI-1640 medium with 2% FBS and added to a 96-wellplate at a concentration of 2×10⁴ cells/well in 75 μl. This wasdesignated as the cell plate.

Peptides were suspended in PPMI-15% FBS (test medium). For the agonistassay, medium was added to rows 2-12 of a 96-well plate. The peptide wasadded to row 1 in 200 μl of test medium at a final concentration of 60μM. The peptide was serially diluted (1:1) across rows 2-11. No peptidewas added to row 12 (control; cells without IGF-1). For the antagonistassay, test medium containing 10 ng/ml IGF-1 (ED₅₀ test medium) wasadded to all wells of a 96-well plate. To row 1 was added 100 μl ofpeptide in ED₅₀ test medium at a concentration of 120 μM. The peptidewas serially diluted (1:1) across rows 2-11. No peptide was added to row12 (control; cells with IGF-1).

For both agonist and antagonist assays, 75 μl from the working plateswas transferred to the appropriate rows in comparable cell plates. Thestarting peptide concentration for both agonist and antagonist assayswas 30 μM. Each peptide was done in duplicate. Plates were incubated at37° C. for 45-48 h. Ten microliters of WST-1 (Cell ProliferationReagent, Roche cat #1 644 807) were added to each well and the plateswere read in an ELISA reader (440/700 dual wavelength) each hour for 4h. Graphs were prepared from the raw data using Sigma Plot. Peptidesincluded:

Site SEQ Pep- IGF- ID tide Formula 1R Sequence NO: 20E2 2 1DYKDFYDAIDQLVRGSARAGGTRD 2209 D815 6 2 WLDQERAWLWCEISGRGCLS 2206 G33 1 1GIISQSCPESFYDWFAGQVSDPWWCW 1600 RP6 2 1 TFYSCLASLLTGTPQPNRGPWERCR 1635RP9 1 1 GSLDESFYDWFERQLG 1559 S175 1 1 GRVDWLQRNANFYDWFVAELG 1560

Results of the IGF-1 proliferation assays are shown in FIGS. 36-42. FIG.36 demonstrates that that peptides G33 (Site 1; ED₅₀˜10 μM) and D815(Site 2; ED₅₀˜2 μM) showed agonist activity at IGF-1R, whereas peptidesRP9 and RP6 showed no agonist activity. FIG. 37 demonstrates that thatpeptides RP6 (Site 1; ED₅₀˜1 μM) and RP9 (Site 1; ED₅₀˜7 μM) showedantagonist activity at IGF-1R, whereas peptides G33 and D815 showed noantagonist activity. FIG. 38 demonstrates that peptides S175 and 20E2exhibited weak agonist activity at IGF-1R (ED₅₀>10 μM). FIG. 39 showsthat D815-RP9 dimers with 6- or 12-amino acid linkers acted as agonistsat IGF-1R. FIG. 40 shows that dimer peptide D815-6-G33 was inactive asan agonist at IGF-1R. FIG. 41 shows that monomer peptide RP6 acted as anantagonist at IGF-1R. The IGF-1 standard curve determined for FDC-P2cells is shown in FIG. 42.

The IGF-1R data for the Site 1 and Site 2 peptides is summarized inTable 15, below.

TABLE 15 Site SEQ Mon./ IGF- ID nM Ki nM ED₅₀ Max nM IC₅₀ Ki/ DimerForm. 1R Link. Sequence NO: app Kd Growth Action Antag. ED50 Class IGF-1NA 0.69 0.30 100 2 2.3 A rG33 1 1 NA GIISQSCPESFYDWFAGQVSDPWWCW 16001450 500 >50 — 2.9 A rD815 6 2 NA WLDQERAWLWCEISGRGCLS 2206 4080500 >50% — 8.2 A RP9 1 1 NA GSLDESFYDWFERQLG 1559 417 — <10% 900 0.5 ND815- 6-1 2-1  6 aa WLDQERAWLWCEISGRGCLSGGSGGSGII 2210 624 — <10% nd ndG33 SQSCPESFYDWFAGQVSDPWWCW D815- 6-1 2-1  6 aaWLDQERAWLWCEISGRGCLSGGSGGSGSL 2211 36 50 >50% >500 0.8 A RP9DESFYDWFERQLGKK D815- 6-1 2-1 12 aa WLDQERAWLWCEISGRGCLSGGSGGSGGS 2212 310,000 100 — 0.0003 A RP9 GGSGSLDESFYDWFERQLGKK A = agonists; N= antagonist; nd = not determined; NA = not applicable; Form. = formula;Mon. = monomer; Antag. = antagonism; Link. = linker; Linker sequencesare underlined.

Example 11 Panning Peptide Libraries for IGF-1 Binding Proteins

A. Panning Secondary Libraries

Soluble IGF-1R (“sIGF-1R”) was obtained from R&D Systems. The solubleprotein (>95% pure) included the heterotetrameric (alpha 2-beta 2)extracellular domain of IGF-1R isolated from a mouse myeloma cell line.sIGF-1R (500 ng/well) was added to an appropriate number of wells in a96-well microtiter plate (MaxiSorp plates, NUNC) and incubated overnightat 4° C. Wells were then blocked with MPBS (PBS buffer pH 7.5 containing2% Carnation® non-fat dry milk) at room temperature (RT) for 1 h. Eightwells were used for each round of panning for the G33 and RP6 secondarylibraries. The phage were incubated with MPBS for 30 min at RT, then 100μl was added to each well.

For the first round, the input phage titer was 4×10¹³ cfu/ml. For rounds2 and 3, the input phage titer was approximately 10¹¹ cfu/ml. Phage wereallowed to bind for 2 to 3 h at RT. The wells were then quickly washed13 times with 200 μl/well of MPBS. Bound phage were eluted by incubationwith 100 μl/well of 20 mM glycine-HCl, pH 2.2 for 30 s. The resultingsolution was then neutralized with Tris-HCl, pH 8.0. Log phase TG1 cellswere infected with the eluted phage, then plated onto two 24 cm×24 cmplates containing 2xYT-AG. The plates were incubated at 30° C.overnight. The next morning, cells were removed by scraping and storedin 10% glycerol at −80° C. For subsequent rounds of affinity enrichment,cells from these frozen stocks were grown and phage were prepared asdescribed above. A minimum of 72 clones was picked at random from thesecond, third, and fourth rounds of panning and screened for bindingactivity. DNA sequencing of the clones determined the amino acidsequences summarized in FIG. 43A-43B.

B. Panning Peptide Dimer Libraries

Microtiter plates were coated and blocked by standard methods, asfollows. Plates were coated with sIGF-1R (see Example, above) or solubleIR (Bass construct; Bass et al., 1996, J. Biol. Chem. 271:19367-19375)in 0.2 M NaHCO₃, pH 9.4. One hundred microliters of solution containingeither 50 ng IR or IGF-1R (rounds 1 and 2), 25 ng IR or IGF-1R (round3), or 12.5 ng IR or IGF-1R (round 4) was added to an appropriate numberof wells in a 96-well microtiter plate (MaxiSorp plates, Nalge NUNC) andincubated overnight at 4° C. Wells were then blocked with a solution of2% non-fat milk in PBS (MPBS) at RT for at least 1 h.

Eight wells coated with IR or IGF-1R were used for each round ofpanning. One hundred microliters of phage were added to each well. Forthe first round, the input phage titer was 3×10¹³ cfu/ml. For subsequentrounds, the input phage titer was approximately 10¹² cfu/ml. Phage wereincubated for 2-3 h at RT. The wells were then quickly washed 13 timeswith 300 μl/well of PBS. Bound phage were eluted by incubation with 150μl/well of 50 mM glycine-HCl, pH 2.0 for 15 min. The resulting solutionwas pooled and then neutralized with Tris-HCl, pH 8.0. Log phase TG1cells were infected with the eluted phage, in 2xYT medium for 1 h at 37°C. prior to the addition of helper phage, ampicillin, and glucose (2%final concentration).

After incubation for 1 h at 37° C., the cells were spun down andresuspended in 2xYT-AK medium. The cells were then returned to theshaker and incubated overnight at 37° C. Phage amplified overnight werethen precipitated and subjected to the next round of panning. A total of96 clones were picked at random from rounds 3 and 4 and screened forbinding activity. Several clones from each pan were further tested forbinding to IR or IGF-1R in phage ELISA by competition with solublepeptides as described in Beasley et al. International ApplicationPCT/US00/08528, filed Mar. 29, 2000, and Beasley et al., U.S.application Ser. No. 09/538,038, filed Mar. 29, 2000. Competition wasperformed by addition of 5 μl of RP9 peptide, recombinant D8 peptide, orboth per well, followed by addition of 100 μl of phage per well.Representative peptides are shown in FIGS. 44A-44B and in Table 16,below.

TABLE 16 SEQ ID Site Pep. NO: Form. IR Sequence Description RP27 22136-1 2-1 GLDQEQAWVECEVYGRGCPYGSLDESFYDWFERQLG No linker RP28 2214 6-1 2-1RLEEEWAWVQCEVYGRGCPSGGSGGSGSLDESFYDWFERQLG EEE Stretch in D8 RP29 22156-1 2-1 SLDREWACVKCEVYGRGCPCGGSGGSGSLDESFYDWFERQLG Repeat isolate RP302216 6-1 2-1 SLEEEWAQVECEVYGRGCPSGGSGGSGSLDESFYDWFERQLG D8 by designRP31 2217 6-1 2-1 SLEEEWAQVECEVYGRGCPSGGSGGSGLLDESFYHWFDRQLR D8 & RP9 bydesign RP32 2218 6-1 2-1 SIEEEWAQIKCDVWGRGCPPGGSGGSGLLDESFYHWFDRQLR D8 &RP9 by design RP33 2219 6-1 2-1 QLDLEWAWVQCEVYGRGCGGSGSLDESFYDWFERQLG 3amino acid linker RP34 2220 6-1 2-1 QLDEEWAGVQCEVYGRGCSLDESFYDWFERQLG Nolinker RP35 2221 6-1 2-1 RLEEEWRWVQCEVYGRGCAAGGSGGSGSLDESFYDWFERQLG EEEStretch in D8 RP36 2222 6-10 2-1SLDQEWAWVQCEVYGRGCPSGGSGGSDSDWAGYEWFEEQLD D8 (W1 −> S)- Group 6 bydesign Pep. = peptide; Form. = formula; Linker sequences are shown inbold and underlined; All dimers are linked C—N

C. Determination of Amino Acid Preferences

For both monomer and dimer peptides, amino acid preferences for eachpeptide were determined as follows. The expected frequency of each ofthe 20 amino acids at that position was calculated based on codon usageand % doping for that library. This was then compared to the actualfrequency of occurrence of each amino acid at every position after fourrounds of biopanning. Any amino acid that occurred at afrequency >2-fold was considered preferred. Most preferred amino acid(s)were those that have the greatest fold enrichment after panning.Preferred amino acid sequences for RP9, D8, and Formula 10 (Group 6)peptides are shown below.

TABLE 17 Peptide Sequence SEQ ID NO: RP9 GSLDESFYDWFERQLG 1559 RegularGLADEDFYEWFERQLR 2223      L w/ Peptide GQLDEDFYEWFDRQLS 2224         Aw/ Insulin GFMDESFYEWFERQLR 2225   W     A

Table 17 shows preferred amino acid sequences for RP9 peptides. Residuesin bold indicate strong preference; underlined residues indicatepositions where more than one amino acid preference is seen. The firstcolumn indicates the conditions used for the panning procedure. “RP9”indicates sequence of the parent RP9; “Regular” indicates regular pan asdescribed in methods for panning of random libraries; “w/ peptide”indicates panning in the presence of 2 nM RP9 peptide; “w/ insulin”indicates panning in the presence of 2 nM insulin.

TABLE 18 SEQ ID Peptide Sequence NO: D8 Parent: WLDQEWAWVQCEVYGRGCPS2129 Dimer Consensus sLEEEWaQIECEVY/WGRGCps 2226 Monomer ConsensussLEEEWaQIqCEIY/WGRGCry 1548            W

Table 18 shows preferred amino acid sequences for D8 peptides. Uppercase residues in bold indicate strong preference (>90% frequency); uppercase letters, non-bold, indicate some preference (5-15% higher frequencythan expected); lower case letters indicate less preference (2-5% higherfrequency than expected); similar preferences seen in D8 in both monomerand dimer libraries. The underlined Y/W indicates that both residues areequally preferred at that position. In the original D8 sequence thatposition is occupied by Y.

TABLE 19 SEQ ID Peptide Sequence Type NO: Group 6 W(A/E)GYEW(F/L)preferred core 1549 Group 6 DSDWAGYEWFEEQLD preferred sequence 1595

Table 19 shows preferred amino acid sequences for Group 6 peptides.Underlined residues indicate preferred N-terminal and C-terminalextensions.

Example 12 Fluorescence-Based hIGF-1R Binding Assays

A. Heterogeneous Time-Resolved Fluorometric Assays

The effect of recombinant peptide G33 (rG33) on the binding ofbiotinylated-recombinant human IGF-1 (b-rhIGF-1) to recombinant humanIGF-1R (rhIGF-1R) was determined using heterogeneous time-resolvedfluorometric assays (TRF; DELFIA®, PE Wallac, Inc.). The rhIGF-1Rprotein included the extracellular domain of the receptorpre-propeptide, up to amino acid residue 932 (A. Ullrich et al., 1986,EMBO J. 5:2503-2512). Duplicate data points were collected at eachconcentration of competitor and the lines were designed to represent thebest fit to a four-parameter non-linear regression analysis(y=min+(max−min)/(1+10̂((log IC₅₀−x)*Hillslope))) of the data, which wasused to determine IC₅₀ values.

The assay was performed using a 96-well clear microplate (NUNC MaxiSorp)with a final volume of 100 μl. Microtiter plates were coated with 0.1 μgrhIGF-1R in 100 μl of NaHCO₃, pH 8.5 buffer, and incubated overnight atroom temperature (RT). The plates were washed 3-times with 0.05 MTris-HCl (pH 8 at 25° C.), 0.138 M NaCl, 0.0027 M KCl (TBS). This wasfollowed by addition of 200 μl blocking buffer (TBS containing 0.05%Bovine Serum Albumin (BSA, Cohn Fraction V)), and incubated for 1 h atRT. The plates were washed 6-times with a 1× solution of Wallac'sDELFIA® wash concentrate. Competitor was added in a volume of 50 μl andserially diluted across the microtiter plate in TBS containing 0.05%BSA. Non-specific binding (background) was determined in the presence of60 μM hIGF-1.

Fifty microliters of b-rhIGF-1, 10 nM, diluted in TBS containing 0.05%BSA was added. The plates were incubated for 2 h at RT. Afterincubation, plates were washed 6-times with a 1× solution of Wallac'sDELFIA® wash concentrate. Then the plates were treated with 100 μL ofWallac's DELFIA® Assay Buffer containing a 1:1000 dilution ofeuropium-labeled streptavidin and incubated for 2 h at RT. This wasfollowed by washing 6-times with a 1× solution of Wallac's DELFIA® washconcentrate. One hundred microliters of Wallac's DELFIA® enhancer wasadded, and the plates were shaken for 30 min at RT. After shaking, thefluorescence signal at 620 nm was read on a Victor² 1420 plate reader(PE Wallac, Inc.). Primary data were background corrected, normalized tobuffer controls, and then expressed as % specific binding. The Z′-factorwas greater than 0.5 (Z′=1−(3σ₊+3σ⁻)/|μ₊−μ⁻|; Zhang et al., 1999, J.Biomol. Screen. 4:67-73) and the signal-to-background (S/B) ratio was˜20. The results of these experiments are shown in FIG. 45. The IC₅₀value calculated for rG33 is shown in Table 20, below.

The effect of recombinant peptides D815 (rD815), RP9, D815-6aa-G33,D815-6aa-RP9, and D815-12aa-RP9 on the binding of b-rhIGF-1 to rhIGF-1Rwas determined using the fluorometric assay described above. IGF-1 wasused as a control. Duplicate data points were collected at eachconcentration of competitor and the lines represent the best fit to afour-parameter non-linear regression analysis, which was used todetermine IC₅₀ values. Results for rD815 are show in FIG. 46; resultsfor RP9 are shown in FIG. 47; results for D815-6-G33 are shown in FIG.48; results for D815-6-RP9 are shown in FIG. 49; and results forD815-12-RP9 are shown in FIG. 50; the results for IGF-1 are shown inFIG. 51. The IC₅₀ values for the rD815, RP9, D815-6aa-G33, D815-6aa-RP9,and D815-12aa-RP9 peptides, and IGF-1 are shown in Table 20, below.Linker sequences are underlined.

TABLE 20 SEQ ID Competitor Sequence NO: IC₅₀ (M) rG33GIISQSCPESFYDWFAGQVSDPWWCW 1600 1.45 × 10⁻⁶ M rD815 WLDQERAWLWCEISGRGCLS2206 4.08 × 10⁻⁶ M RP9 GSLDESFYDWFERQLG 1559 4.17 × 10⁻⁷ M D815-6aa-G33WLDQERAWLWCEISGRGCLSGGSGGSGIIS 2210 6.24 × 10⁻⁷ M QSCPESFYDWFAGQVSDPWWCWD815-6aa-RP9 WLDQERAWLWCEISGRGCLSGGSGGSGSL 2211 3.57 × 10⁻⁸ MDESFYDWFERQLGKK D815-12aa-RP9 WLDQERAWLWCEISGRGCLSGGSGGSGG 2212 3.22× 10⁻⁹ M SGGSGSLDESFYDWFERQLGKK IGF-1 6.85 × 10⁻¹⁰ M

The order of potency of all peptides or dimers compared to IGF-1 wasdetermined as:IGF-1>D815-12aa-RP9>>D815-6aa-RP9>RP9≅D815-6aa-G33>rG33>rD815. Theseresults suggest that the coupling of D815 with RP9 using an extendedlinker (12 versus 6 amino acids) produced a potent competitor thatapproximates the affinity of IGF-1 for its own receptor.

B. Time-Resolved Fluorescence Resonance Energy Transfer Assays

The effect of Site 1 peptides, Site 2 peptides, and rhIGF-1 on thedissociation of biotinylated-20E2 (b-20E2, Site 1) from recombinanthuman IGF-1R was determined using time-resolved fluorescence resonanceenergy transfer assays (TR-FRET). Best fit non-linear regressionanalysis of the data, was used to determine dissociation rate constants.Each data point represents a single observation.

The assay was performed using a 96-well white microplate (NUNC) with afinal volume of 100 μl. Final incubation conditions were 16.5 nM b-20E2,2.2 nM SA-APC (streptavidin-allophycocyanin), 2.2 nM Eu³⁺-rhIGF-1R(LANCE™ labeled, PE Wallac, Inc.), 0.05 M Tris-HCl (pH 8 at 25° C.),0.138 M NaCl, 0.0027 M KCl, and 0.1% BSA (Cohn Fraction V). Reactionswere allowed to reach equilibrium for 6 h at RT. Following this, variouspeptides or IGF-1 were added at a final concentration of 100 μM or 30μM, respectively. The addition of peptides or IGF-1 initiated themeasurement of dissociation (Time Zero, sec). The fluorescence signal at665 nm was read on a Victor² 1420 plate reader (PE Wallac, Inc.) at 30sec intervals.

Results of these experiments are shown in FIG. 52. The buffer controlsdid not vary over the time interval of study, which demonstrated thatthe equilibrium was not disturbed by the addition of diluent at Timezero. The addition of excess (>1000-fold 20E2 K_(d) for IGF-1R) Site 1peptides such as H2C, 20E2, or RP6 did not differ depending on specificthe peptide used, and the dissociation rates of b-20E2 were similar forthese peptides. D8B12 (Site 2 peptide) and IGF-1 (binds both Site 1 andSite 2) did demonstrate significant differences in the rate ofdissociation of b-20E2. This would suggest that these agents act asnon-competitive or allosteric regulators of Site 1 binding.

The effect of various peptides or peptide dimers on the binding ofbiotinylated-20E2 (B-20E2) to recombinant human IGF-1R was determinedusing TR-FRET assays, described above. For these experiments, each datapoint represents the average of two replicate wells. The lines representthe best fit to a four-parameter non-linear regression analysis(y=min+(max−min)/(1+10̂((log IC₅₀-x)*Hillslope))) of the data, which wasused to determine IC₅₀ values.

The assays were performed using a 384-well white microplate (NUNC) witha final volume of 30 μl. Final incubation conditions were 15 nM b-20E2,2 nM SA-APC, 2 nM Eu³⁺-rhIGF-1R (LANCE™ labeled, PE Wallac, Inc.), 0.05M Tris-HCl (pH 8 at 25° C.), 0.138 M NaCl, 0.0027 M KCl, and 0.1% BSA(Cohn Fraction V). After 16-24 h of incubation at RT, the fluorescencesignal at 665 nm and 620 nm was read on a Victor² 1420 plate reader (PEWallac, Inc.). Primary data were background corrected, normalized tobuffer controls, and then expressed as % specific binding. The Z′-factorwas greater than 0.5 (Z′=1−(3σ₊+3σ⁻)/|μ₊−μ⁻|; Zhang et al., 1999, J.Biomol. Screen. 4:67-73) and the signal-to-background (S/B) ratio was˜4. Results of these experiments are shown in FIG. 53. Table 21, below,shows the IC₅₀ values calculated for these experiments. Notably, the C1peptide showed IGF-1R affinities of ˜1 nM (FIG. 53) and ˜10 nM (Table21) in these assays.

TABLE 21 SEQ ID Site Competitor Sequence NO: Formula IGF-1R IC₅₀ (M) C1CWARPCGDAANFYDWFVQQAS 1550 1 1 8.80E−10 IGF-1 2.93E−09 RP9GSLDESFYDWFERQLG 1559 1 1 3.93E−08 20E2 DYKDFYDAIDQLVRGSARAGGTRD 2209 21 1.04E−07 E8 GGTVWPGYEWLRNA 2118 10 2 2.53E−07 H2C FHENFYDWFVQRVSKK2117 1 1 4.60E−07 S173 LDALDRLMRYFEERPSL 1830 3 1 6.29E−06 D8B12WLEQERAWIWCEIQGSGCRA 1884 6 2 1.13E−05 A6 SAKNFYDWFVKK 1551 1 1 3.10E−05

C. Fluorescence Polarization Assays

The effect of various peptide monomers and dimers on the binding offluorescein-RP9 (FITC-RP9) to soluble human insulinreceptor-immunoglobulin heavy chain chimera (sIR-Fc; Bass et al., 1996,J. Biol. Chem. 271:19367-19375) was determined using fluorescencepolarization assays (FP). For these experiments, each data pointrepresents the average of two replicate wells. The lines represent thebest fit to a four-parameter non-linear regression analysis of the data,which was used to determine IC₅₀ values.

The assays were performed in a 384-well black microplate (NUNC) with afinal volume of 30 μl. Final incubation conditions were 1 nM FITC-RP9,10 nM sIR, 0.05 M Tris-HCl (pH 8 at 25° C.), 0.138 M NaCl, 0.0027 M KCl,0.05% BGG (bovine gamma globulin), 0.005% Tween-20. After 16-24 h ofincubation at RT, the fluorescence signal at 520 nm was read on anAnalyst™ AD plate reader (LJL BioSystems, Inc.). Primary data werebackground corrected using 10 nM sIR without FITC-RP9 addition,normalized to buffer controls and then expressed as % specific binding.The Z′-factor was greater than 0.5 (Z′=1−(3σ₊+3σ⁻)/|μ₊−μ⁻|; Zhang etal., 1999, J. Biomol. Screen. 4:67-73) and the assay dynamic range was˜125 mP. In parallel with these experiments, TR-FRET assays wereperformed using rhIGF-1R and b-20E2, as described above. Results of theFP and TR-FRET experiments are shown in Table 22, below.

TABLE 22 FP TR-FRET Bndg Ratio Site SEQ ID Peptide sIR-Fc rhIGF-1RIGF-1R/1R Form. IGF-1R NO: Sequence RP4   17 8100 476 2 1 1552PPWGARFYDAIEQLVFDNL S175   10 1650 165 1 1 1560 GRVDWLQRNANFYDWFVA ELGRP15   28  706 25 1 1 2130 SQAGSAFYAWFDQVLRTV H2C(D117)   66  600 9 1 12117 FHENFYDWFVQRVSKK 20E2(D118)   51  100 1.9 2 1 2209DYKDFYDAIDQLVRGSARA GGTRD RP9   24   33 1.4 1 1 1559 GSLDESFYDWFERQLGG33  139  178 1.3 1 1 1600 GIISQSCPESFYDWFAGQV SDPWWCW E8(D120)  206 175 0.85 10 2 2118 GGTVWPGYEWLRNA C1(D112)   52   10 0.19 1 1 1550CWARPCGDAANFYDWFV QQAS RP16 6400  961 0.15 1553 VMDARDDPFYHKLSELVT FPsIR-Fc column shows IC₅₀ (nM) values obtained (vs. FITC-RP9); TR-FRETrhIGF-1R column shows IC₅₀ (nM) values obtained (vs. b-20E2); forbinding ratio: higher values indicated higher affinity for IR thanIGF-1R. Form. = formula; Bndg. = binding.

These results demonstrated that S175, RP4, and RP15 showed highaffinities for IR and showed high binding ratios for IGF-1R over IR.H2C, 20E2, RP9, and C1 were slightly less potent than S175, RP4, andRP15 at IR, and these peptides had lower binding ratios for IGF-1R overIR. G33 and E8 were less potent than S175, RP4, and RP15 at IR, andshowed comparable binding to IGF-1R and IR. RP16 had poor potency at IRand IGF-1R, but had higher affinity for IGF-1R than IR.

Example 13 IGF-1R Binding Peptides—Additional Isolates

The isolation and characterization of peptides which bind to andsubdivide the insulin receptor binding site into multiple,non-overlapping regions designated Site 1 and Site 2 has been previouslydescribed (Beasley et al., U.S. application Ser. No. 09/538,038, filedMar. 29, 2000, published as WO 01/72771; Pillutla et al., U.S. patentapplication Ser. No. 09/962,756, filed Sep. 24, 2001; Pillutla et al.,2002, J. Biol. Chem. 277:22590-22594). To identify IGF-1R antagonists, amulti-tiered approach was used. First, Site 1 peptides with greaterselectivity for IGF-1R as compared to IR were identified. Second,secondary libraries were generated using information from the primarylibrary pannings. These secondary libraries were designed to define theamino acid requirements for binding, specificity, and affinity.

To determine optimal sequence requirements within the motif, a secondarylibrary based on a clone identified from the random library was madewhere the flanking regions were held constant, while the core wasallowed to change. The library was prepared from doped oligonucleotidesso that half of the amino acid residues (on average) in the coresequence were altered per peptide. Panning of these libraries identifiedsubstitutions within the core that did or did not affect binding. In analternative approach, amino acids in the flanking regions conferringbinding affinity and/or specificity were defined by designing secondarylibraries wherein the core was held constant and the flanking sequenceswere either doped or randomized. For both types of libraries, aminoacids optimal for binding were selected by panning against IGF-1R. Oncesecondary peptides with the appropriate binding characteristics wereidentified, a preferred peptide was defined. To do this, the amino acidsat each position were optimized based on a comparison of the expectedresults from the doping strategy and the actual results observed in thebinding population.

A. Primary Peptide Libraries

The E. coli, strain TG1 (genotype=K12Δ(lac-pro), supE, thi,hsdΔ5/F′[traD36, proAB, lacI^(q), lacZΔM15]) was obtained from Pharmacia(Piscataway N.J.). DNA fragments coding for peptides containing 40random amino acids were generated by a PCR-technique using syntheticoligonucleotides. A 145-base oligonucleotide was synthesized to includethe sequence (NNK)₄₀ where N=A, C, T, or G and K=G or T. Thisoligonucleotide was used as the template in PCR reactions along with twoshorter oligonucleotide primers, both of which were biotinylated attheir 5′ ends. The resulting product was purified, concentrated, andligated to the phagemid pCANTAB5E (Pharmacia). The ligation product waspurified and electroporated into competent bacterial cells. Thetransformants were grown at 37° C. for 1 h, pooled and plated ontoselection medium. Depending upon the amount of DNA electroporated, thediversity of the random 40mer peptide cell library was found to bebetween 1.6×10¹⁰ and 1×10¹¹ independent clones. The phage library wasproduced by rescue of the cell library according to standard phagepreparation protocols (G. P. Smith and J. K. Scott, 1993, MethodsEnzymol. 217:228-257). Phage titers were usually at 4×10¹³ cfu/ml. Inprevious experiments, sequencing of randomly selected clones from thecell library indicated that about 54% of all clones were in-frame. Theshort FLAG sequence (DYKD; SEQ ID NO:1545), was included at theN-terminus as an immunoaffinity tag. In addition, the E-tag epitope(GAPVPYPDPLEPR; SEQ ID NO:XX) was engineered into the carboxy terminusof the peptide. Additional random phage libraries of 20mer peptides wereconstructed using a similar approach. The diversity of these celllibraries was estimated to be >1.1×10¹¹ clones.

B. Secondary and Tertiary Libraries

The desired number of amino acid mutations were introduced in theparental peptide at the codon level when the synthetic DNA template wasproduced. For example, where a change in 45% of the amino acids wasdesired (i.e., 9 changes/20 amino acids), then a 60% change at the codonlevel was needed due to the redundancy of the genetic code (efficiencyfactor of 0.75). Per position, this translated to 20% doping at thelevel of DNA synthesis. At the DNA synthesis level, a 20% dopingincluded the following ratio of nucleotides in the synthetic template:

A  80% A, 6.6% C, 6.6% G, 6.6% T C 6.6% A  80% C, 6.6% G, 6.6% T G 6.6%A 6.6% C,  80% G, 6.6% T T 6.6% A 6.6% C, 6.6% G,  80% T

In this chart, the A, C, T, G (underlined and in bold) bases representthe original bases in the parental sequence. When the clones from celllibraries were sequenced and the number of amino acid mutations wasexamined per peptide, the average number of changes was found tocorrelate to the desired value. After the synthetic template wasobtained, the DNA was ligated to the pCANTBA5E phagemid vector toproduce the cell library in the TGI strain as previously described.Phage rescue was carried out to produce the phage library used in thepanning experiments.

C. Panning of peptide libraries

A standard method was used to coat and block all microtiter plates.Plates were coated with IGF-1R in 0.2 M NaHCO₃, pH 9.4. One hundredmicroliters of solution containing 100 ng of IGF-1R was added to anappropriate number of wells in a 96-well microtiter plate (MaxiSorpplates, Nunc) and incubated overnight at 4° C. Wells were then blockedwith a solution of 2% non-fat milk in PBS (MPBS) at room temperature(RT) for at least 1 h.

Four to eight wells coated with IGF-1R were used for each round ofpanning. One hundred microliters of phage were added to each well. Forthe first round, the input phage titer was ˜10¹³ cfu/ml. For subsequentrounds, the input phage titer was approximately 10¹² cfu/ml. Phage wereallowed to bind for 2-3 h at RT. The wells were then quickly washed 13times with 300 μl/well of PBS. Bound phage were eluted by incubationwith 150 μl/well of 50 mM glycine-HCl, pH 2.0 for 5 min. The resultingsolution was pooled and then neutralized with Tris-HCl, pH 8.0.

Log phase TG1 cells were infected with the eluted phage, in 2xYT mediumfor 1 h at 37° C. prior to the addition of helper phage, ampicillin andglucose (2% final concentration). After incubation for another hour at37° C., the cells were spun down and resuspended in 2xYT-AK medium. Thecells were then returned to the shaker and incubated overnight at 37° C.Phage amplified overnight was then precipitated and subjected to thenext round of panning. A total of 96 clones were picked at random fromrounds 3 and 4 and screened for binding activity.

D. ELISA Analyses of Phage

For phage pools, cells from frozen stocks were grown and phage wereprepared as described above. For analysis of individual clones, colonieswere picked and phage prepared as described above. Subsequent steps werethe same for pooled and clonal phage. Microtiter wells were coated andblocked as described above. Wells were coated with either IGF-1R or IR.Phage resuspended in MPBS (PBS containing 2% non-fat milk) were added towells (100 μl/well) and incubated at room temperature for 1 h. The phagesolution was then removed, and the wells were washed three times withPBS at room temperature.

Anti-M13 antibody conjugated to horseradish peroxidase (PharmaciaBiotech) was diluted 1:3000 in MPBS and added to each well (100μl/well). Incubation was for another hour at room temperature, followedby PBS washes as described. Color was developed by addition of ABTSsolution (100 μl/well; Boehringer). Color development was stopped byadjusting each well to 0.5% SDS. Plates were analyzed at 405 nm using aSpectraMax 340 plate reader (Molecular Devices) and SoftMax Prosoftware. Data points were averaged after subtraction of appropriateblanks. A clone was considered “positive” if the A₄₀₅ of the well was≧2-fold over background.

E. Determination of Amino Acid Preferences

Amino acid preferences for each peptide were determined as follows. Theexpected frequency of each of the 20 amino acids at that position wascalculated based on codon usage and % doping for that library. This wasthen compared to the actual frequency of occurrence of each amino acidat every position after four rounds of biopanning. Any amino acid thatoccurred at a frequency ≧2-fold was considered preferred. The mostpreferred amino acid(s) were defined as those with the greatestenrichment after panning. Using the amino acid preferences determinedfor each position, peptides with the most preferred sequences weredesigned.

Representative monomer and dimer peptides identified by panningsecondary libraries for binding to IGF-1R are shown in FIGS. 54A-54B,55A-55B, 56A-56B, 57A-57B, 58A-58B, 59A-59B, 60A-60C, 61A-61B, 62A-62B,63A-63B, and 64A-64B. Primary library pannings produced severalpeptides, including RP6, RP48, RP52, RP54, RP56, and RP60, describedabove. Peptides designed according to amino acid preferences (i.e.,peptides by design) included RP30-IGF, RP31-IGF, and RP33-IGF.

Example 14 IGF-1 Antagonist Peptides

A. Cells and Reagents

MCF-7 and MiaPaCa cell lines were obtained from the American TypeCulture Collection (Manassas, Va.). Cells were routinely grown inRPMI1640 medium supplemented with 10% fetal bovine serum and 1%glutamax. The extra-cellular domain of IGF-1R was obtained as arecombinant protein from R&D Systems (Minneapolis, Minn.).

B. Whole-Cell Lysates

For qualitative IRS-1 phosphorylation analysis, MCF-7 cells in monolayercultures (about 80% of confluency) were used. After about 20 h ofstarvation in serum-free RPMI medium (GibcoBRL), cells were stimulatedfor 10 min in the same medium containing IGF-1 (Peprotech), or IGF-1plus peptides (synthetic peptides produced by Research Genetics), or noaddition as a negative control. After treatment, cells were rinsed twicewith ice-cold PBS containing 0.2 mM PMSF and 1 mM Na₃VO₄ (all fromSIGMA). Cells were scraped into the same buffer and pelleted bycentrifugation at 200×g for 3 min. Lysis was done in RIPA buffer(0.8766% NaCl, 0.11% SDS, 0.5% deoxycholic acid (all from SIGMA), 1%Triton X-100, (Boehringer Mannheim)) containing phosphatase inhibitorcocktails 1 and 2 (SIGMA) and protease cocktail inhibitor tablet(Boehringer Mannheim) for 5 min on ice. Cell lysates were cleared bycentrifugation for 5 min at 14 000×g and the resulting supernatant wassnap-frozen in ethanol-dry ice and stored at −80° C. The proteinconcentration was determined using the D_(C) Protein Assay Kit (Bio-RadLaboratories).

C. Immunoprecipitation and Western Blot Analysis

Immunoprecipitations were performed with pre-cleared lysates for 4 h at40° C. using 0.3-0.5 mg total protein with 1 μg polyclonal anti-IRS-1antibody (Upstate Biotechnology), and 25 μl protein A/agarose slurry(SIGMA). Agarose beads with immobilized proteins were washed 3 timeswith IP wash buffer (50 mM Tris pH 7.5 (GibcoBRL), 150 mM NaCl, 1 mMNa₃VO₄, 0.2 mM PMSF). Protein elutions and denaturation were done for 3min at 95° C. in 30 μl of Laemmle sample buffer (Bio-Rad Laboratories)with 0.5 M β-mercaptoethanol (SIGMA).

Immunoprecipitates were subjected to SDS-PAGE on 4-15% Tris-HCl ReadyGels and transferred to Trans-Blot Transfer Medium nitrocellulosemembranes (both from Bio-Rad Laboratories). Membranes were blocked withPBS-Tween 20 (SIGMA) containing 2% non-fat milk. For detection of IRS-1protein, blots were incubated with anti-IRS-1 antibody, followed bysecondary antibody goat anti-rabbit IgG, HRP-conjugate. For detection ofphosphorylated IRS-1, blots were incubated with monoclonalanti-phosphotyrosine (4G10) HRP-conjugated antibody. All antibodies wereobtained from Upstate Biotechnology. Blots were exposed to an enhancedchemifluorescence substrate (ECL Western Blotting Analysis System,Amersham Pharmacia Biotech). Films were developed and fluorescent signalwas visualized for qualitative analysis.

D. MCF-7 and MiaPaCa Cell Assays

Peptides produced synthetically were maintained as 30 mM stock in 100%DMSO, while recombinant dimers were diluted in water. All synthetic andrecombinant peptides were stored at −80° C. The final concentration ofDMSO was <0.1%. MCF-7 and MiaPaCa (ATCC, Rockville, Md.) cells weremaintained in RPMI containing 10% FBS. All cells were starved overnightby growing them in RPMI media, which was serum free. Cells weretrypsinized, washed twice with PBS before being seeded at 1-3×10³ cellsper well in a 96-well plate with a volume of 150 μl/well. All pointswere done in duplicate in 96-well plates. For antagonist activityassays, immediately before the addition of peptides, all media wasgently removed from the wells. Peptides were serially diluted 1:2 in afinal volume of 150 μl in a separate plate using RPMI containing 0.1%FBS plus 50 nM IGF-1. This mixture was transferred onto the cells, andthe plates were incubated for 72 h at 37° C. in a CO₂ incubator. Toquantitate cell number, 10 μl of WST-1 reagent (Roche MolecularBiochemicals, Indianapolis, Ind.) was added to each well and the plateswere returned to the 37° C./CO₂ incubator for approximately 2 h.Measurements were then read at 440 nm, with 700 nm used as a reference.

E. Binding (ALPHAScreen) Assays

To assay binding, the relative potencies of peptides as compared toIGF-1 were analyzed in a competition system utilizing biotinylated-humanIGF-1 (b-hIGF-1) and His-tagged soluble recombinant human IGF-1R(srhIGF-1R-his; R&D systems, Inc., Minneapolis, Minn.). Detection of thereceptor ligand interaction was measured in an amplified luminescentproximity homogeneous assay (ALPHAScreen; BioSignal-Packard, Montreal).The assay was performed in 384-well Nunc™white polystyrene microplates(Nalge Nunc International, Naperville, Ill.) with a final volume of 40μl. Final incubation conditions were 1 nM b-hIGF-1, 10 nM srhIGF-1R-his,0.025 M HEPES (pH 7.4 at 25° C.), 0.100 M NaCl, 0.1% BSA (Cohn FractionV; Sigma Chemical Co., St. Louis, Mo.), 10 μg/ml nickel conjugatedacceptor beads, and 10 μg/ml streptavidin conjugated donor beads.

For the first step of the assay, hIGF-1 (PeproTech, Inc., Rocky Hill,N.J.), b-hIGF-1 (see below), and peptides were incubated for 2 h at roomtemperature. Each concentration of competitor was assayed in duplicate.Non-specific binding was determined in the presence of 3×10⁻⁵ M hIGF-1.In the second step of the assay, acceptor beads were added and theincubation was continued for 0.5 h. In the final step, donor beads wereadded and the incubation was continued for an additional 1 h. At the endof the incubation period, the fluorescence signal at 520 nm was read ona Fusion-α HT plate reader (Packard BioScience Company, Meriden, Conn.).Primary data were background corrected, normalized to buffer controls,and then expressed as % specific binding. The data were fit to afour-parameter non-linear regression analysis(y=min+(max−min)/(1+10̂((log IC50−x)*Hillslope))), which was used todetermine IC₅₀ values. The Z′-factor for this assay was greater than 0.7(Z′=1-(3σ++3σ−)/|μ+−μ−|) and the signal-to-background (S/B) ratio wasbetween 40 and 70.

Human IGF-1 was biotinylated on free amino groups using Pierce EZ-Link™Sulfo-NHS-LC-Biotinylation Kit (PN #21430, Pierce, Rockford, Ill.).Human IGF-1, at 2 mg/ml in PBS, pH 7.2, was incubated at roomtemperature for 30 min with a 20-fold excess of sulfo-NHS-LC-biotin overtheoretical total free amino groups. Unreacted biotins were removed byextensive dialysis (Pierce Slide-A-Lyzer® Dialysis Cassettes) againstPBS, and degree of conjugation was determined by HABA(2-(4′-hydroxyazobenzene)benzoic acid) assay (Pierce product literature#21430). Number of biotins per hIGF-1 varied between 3 and 5.

F. FDC-P2 Cell Assays

Peptides produced synthetically were maintained as 30 mM stock in 100%DMSO, while recombinant dimers were diluted in water. All synthetic andrecombinant peptides were stored at −80° C. The final concentration ofDMSO was <0.1%. FDC-P2 (obtained from Dr. J. Pierce, National Institutesof Heath, Bethesda, Md.) cells were maintained in RPMI containing 15%FBS and 5% WEHI (Genoquest, Germantown, Md.) at 37° C. in a CO₂incubator. To initiate experiments, all cells were starved for 5 h inRPMI containing 1% FBS. Cells were seeded at 1×10⁴ cells per well into96-well plates at a volume of 75 μl/well. Peptides were added at 2×final concentrations and all points were done in duplicate. Forantagonist assays, peptides at 2× concentration were serially diluted1:2 in a final volume of 75 μl in a separate plate using RPMI containing0.1% FBS and 1 nM IGF-1. This mixture was transferred onto the cells toyield a final volume of 150 μl. The plates were incubated for 48 h at37° C. in a CO₂ incubator. To quantitate cell number, 10 μl of WST-1reagent (Roche Molecular Biochemicals, Indianapolis, Ind.) was added toeach well and the plates were returned to the 37° C./CO₂ incubator forapproximately 2 h. Measurements were then taken at 440 nm, with 700 nmused as a reference.

G. Results

Peptide RP33-IGF exhibited an affinity for IGF-1R close to that of IGF-1(9 nM; Table 23). Other peptides, such as RP54 showed affinity in themicromolar range (Table 23). In contrast to the observations made forIR, competition experiments indicated that IGF-1R Site 1 and 2 peptideswere able to compete with each other. This suggested that the functionalinteractions between Site 1 and Site 2 in IGF-1R differed from thosefound in IR (unpublished data).

To determine if any Site 1 peptides could act as antagonists,proliferation assays were established utilizing IGF-1 and IGF-2responsive human tumor cell lines. Sixteen human tumor cell lines werescreened for their ability to proliferate in the presence IGF-1 andIGF-2 under serum-free conditions. Two cell lines, MCF-7 (breastcarcinoma) and MiaPaCa (pancreatic carcinoma), showed the best doseresponse curves for IGF-1 (ED₅₀=5 nM; FIGS. 65A-65F), and were used forsubsequent experiments.

Peptides were synthesized and screened in the proliferation assay at anIGF-1 dose ten times the ED₅₀ (50 nM). Several antagonist peptides wereidentified, including RP33-IGF, which consistently blocked IGF-1 andIGF-2 proliferation of both MCF-7 and MiaPaCa (FIGS. 66B-66C). Inaddition, peptides RP52 and RP54 were found to act as antagonists in atleast one cell line (Table 26; FIGS. 70A-70B). Peptides RP52 and RP54are classified as miscellaneous peptides, which were not categorizedinto any of the formulae (e.g., Formula 1, Formula 2, etc.) disclosedherein.

Experiments were then performed to determine whether antagonist peptidescould block receptor activation at the level of key signalingintermediate, IRS-1. First, the optimal time and concentration of IGF-1needed for maximal activation of IRS-1 was established (FIGS. 67A-67Band FIGS. 68A-68B). Maximum phosphorylation of IRS-1 was observed after10 min of treatment and was followed by a drop-off of the signal (FIGS.67A-67B). This pattern was presumably due to degradation of the IRS-1protein by a mechanism involving proteasomes (Lee et al., 2000, Mol.Cell. Biol., 2000, 20:1489-1496). Second, RP33-IGF was compared to twounrelated peptides. The RP33-IGF peptide inhibited IRS-1phosphorylation, whereas the unrelated peptides had no effect in theproliferation assay (FIGS. 69A-69B).

The RP6KK peptide was also tested for activity, since the RP33-IGFpeptide was originally derived from the RP6KK sequence. Both RP6KK andRP33-IGF were found to effectively block activation of IRS-1 by IGF-1(FIGS. 69A-69B). At the concentration used, greater than 90% of theprotein was unphosphorylated, indicating that both peptides efficientlyblocked IGF-1R activation. However, RP33-IGF differed from RP6KK by 11amino acids, and RP33-IGF was a superior IGF-1R antagonist in the cellproliferation assays (Tables 24-25). The difference in biologicalactivity did not appear to be related to stability of the peptides sinceboth were found to remain intact during the course of the assays(unpublished data).

TABLE 23 Pep. (Clone) Sequence Site* Formula Affin. (μM) Activ.^(&) IC₅₀(μM) RP33-IGF

1 2 0.009 Antag. 0.1 - MCF-70.7 - MiaPaCa RP6KK

1 2 0.19 Antag. RP52(20C-3-A3-IGFR)

Misc. ND Antag. 0.5 - MCF-7 RP54(20C-4-A7-IGFR)

Misc. 1.6 Antag. 2.5 - MCF-74.3 - MiaPaCa Peptide antagonists of IGF-1Ridentified from primary and secondary library pannings. *site to which apeptide binds was assigned based on competition assays using both IR andIGF-1R as target; ⁺affinity was determined using the AlphaScreen assayversus IGF-1 as described herein; ^($)antagonism was determined from theproliferation assays in the presence of IGF-1 using MCF-7 and MiaPaCacells as described herein; ND = not done; Pep. = peptide; Affin. =affinity; Antag. = antagonist; Misc. = miscellaneous peptide; Cysteineresidues are boxed.

TABLE 24 IGF-1R Antagonists in MCF-7 Cells Site Cellular Binding PeptideForm. IGF-1R IC₅₀ (M) IC₅₀ (M) Sequence H2C-A-H6 1 1 4.0E−07 3.2E−05

C1KK 1 1 2.8E−06 4.2E−08

RP33K-IGF 2 1 2.1E−08 1.8E−09

RP6KK 2 1 9.0E−06 1.8E−07

RP54 Misc. 4.3E−06 4.9E−07

RP52 Misc. 4.5E−07 3.0E−05

RP30-IGF-12-RP30-IGF 2-2 1-1 3.4E−06 2.4E−07

D8B12-12-RP9 6-1 2-1 6.9E−06 N/A

Monomer and dimer peptides which block IGF-1 activity in cellproliferation assays in MCF-7 cells. Form. = formula; Misc. =miscellaneous sequence; Cysteine pairs are shaded and underlined; FY,WF, and L residues from Formula 1 and Formula 2 motifs are shaded andshown in bold.

TABLE 25 IGF-1R Antagonists in MiaPaCa Cells Site Cellular BindingPeptide Form. IGF-1R IC₅₀ (M) IC₅₀ (M) Sequence RP30-IGF 2 1 2.0E−061.6E−07

RP43 2 1 1.0E−07 1.9E−07

RP33K-IGF 2 1 2.0E−07 1.8E−09

L-RP9ex 1 1 1.3E−05 2.2E−06

RP54 Misc. 5.9E−06 4.9E−07

RP56 Misc. 9.4E−06 6.0E−05

RP30-IGF-12-RP30-IGF 2-2 1-1 4.6E−06 2.4E−07

D8B12-12-RP9 6-1 2-1 4.7E−05 N/A

Monomer and dimer peptides which block IGF-1 activity in cellproliferation assays in MiaPaCa cells. Form. = formula; Misc. =miscellaneous sequence; Cysteine pairs are shaded and underlined; FY,WF, and L residues from Formula 1 and Formula 2 motifs are shaded andshown in bold.

TABLE 26 IGF-1R Antagonists in FDC-P2 cells Site Cellular BindingPeptide Form. IGF-1R IC₅₀ (M) IC₅₀ (M) Sequence RP30-IGF 2 1 4.0E−061.6E−07

RP9-lig 1 1 3.2E−04 2.2E−07

lig-RP9 1 1 8.8E−08 1.5E−06

RP43 2 1 7.0E−06 1.9E−07

H2C-A-H6 1 1 3.0E−07 3.2E−05

RP6KK 2 1 1.0E−06 N/A

C1 1 1 4.0E−06 4.2E−08

RP33-IGF 2 1 7.0E−06 1.9E−06

RP6 2 1 5.0E−06 3.5E−07

RP9 1 1 2.0E−06 9.7E−07

RP9-RP9 (C-C) 1-1 1-1 3.0E−05 1.2E−07

RP9-RP9 (C-N) 1-1 1-1 3.0E−05 1.7E−07

G33-RP9 1-1 1-1 1.0E−06 N/A

RP9-L-RP9 1-1 1-1 9.0E−07 3.4E−06

RP9-L-RP6 1-2 1-1 3.0E−06 N/A

G33-D8B12 1-6 1-2 3.0E−06 N/A

D8B12-RP9 6-1 2-1 1.0E−05 N/A

Monomer and dimer peptides which block IGF-1 Activity in cellproliferation assays in FDC-P2 cells. Form. = formula; Lig =Diaminopropionic acid with a 2-aminohydroxyacetyl group (CO—CH2—O—NH2)on the side chain amino group; Numbers such as 17, 19, 12, representspecific chemical linkers (see Table 3); C-C = C-terminal to C-terminallinkage; N-N = N-terminal to N-terminal linkage; Cysteine pairs areshaded and underlined; FY, WF, and L residues from Formula 1 and Formula2 motifs are shaded and shown in bold.

Example 15 IGF-1 Agonist Peptides

A. MCF-7 and MiaPaCa Cell Assays

Peptides produced synthetically were maintained as 30 mM stock in 100%DMSO, while recombinant dimers were diluted in water. All synthetic andrecombinant peptides were stored at −80° C. The final concentration ofDMSO was <0.1%. MCF-7 and MiaPaCa (ATCC, Rockville, Md.) cells weremaintained in RPMI containing 10% FBS. All cells were starved overnightby growing them in serum-free RPMI media. Cells were trypsinized, washedtwice with PBS before being seeded at 1-3×10³ cells per well in a96-well plate in a volume of 150 μl/well. All points were done induplicate in 96-well plates. For agonist activity assays, immediatelybefore the addition of peptides, all media was gently removed from thewells. Peptides were serially diluted 1:2 in a final volume of 150 μl ina separate plate using RPMI containing 0.1% FBS. The diluted peptidesolutions were transferred onto the cells, and the plates were incubatedfor 72 h at 37° C. in a CO₂ incubator. To quantitate cell number, 10 μlof WST-1 reagent (Roche Molecular Biochemicals, Indianapolis, Ind.) wasadded to each well and the plates were returned to the 37° C./CO₂incubator for approximately 2 h. Measurements were then taken at 440 nm,with 700 nm used as a reference.

B. FDC-P2 Cell Assays

Peptides were maintained and stored as indicated above. FDC-P2 cells(obtained from Dr. J. Pierce, NIH) were maintained in RPMI containing15% FBS and 5% WEHI (Genoquest, Germantown, Md.) at 37° C. in a CO₂incubator. To initiate experiments, all cells were starved for 5 h inRPMI containing 1% FBS. Cells were seeded at 1×10⁴ cells per well into96-well plates at a volume of 75 μL/well. Peptides were added at 2×final concentration and all points were done in duplicate. For agonistassays, peptides at 2× concentration were serially diluted 1:2 in afinal volume of 75 μl in a separate plate using RPMI containing 0.1%FBS. The diluted peptide solutions were transferred onto the cells toyield a final volume of 150 μl. The plates were incubated for 48 h at37° C. in a CO₂ incubator. To quantitate cell number, 10 μl of WST-1reagent (Roche Molecular Biochemicals, Indianapolis, Ind.) was added toeach well and the plates were returned to the 37° C. incubator forapproximately 2 h. Measurements were taken at 440 nm, with 700 nm usedas a reference.

For these experiments, potencies of peptide competition were determinedusing the AlphaScreen assay format. Primary data were backgroundcorrected, normalized to buffer controls and then expressed as %specific binding. The data were fit to a four-parameter non-linearregression analysis (y=min+(max−min)/(1+10̂((log IC₅₀−x)*Hillslope))),which was used to determine IC₅₀ values. The Z′-factor for this assay isgreater than 0.7 (Z′=1−(3σ₊+3σ⁻)/|μ₊−μ⁻|) and the signal-to-background(S/B) ratio was between 40 and 70.

C. Results

Several IGF-1R agonist peptides were identified which consistentlystimulated proliferation of both MCF-7 and MiaPaCa cells (Tables 27-28;FIGS. 73A-73D and FIGS. 74A-741). Monomer peptides with IGF-1R agonistactivity included RP60, RP48, G33, C1, and L-RP9ex (Tables 27-28). Dimerpeptides with IGF-1R agonist activity included RP30-IGF-12-D112,RP30-IGF-12-RP31-IGF, RP31-IGF-12-RP30-IGF, D112-12-RP30-IGF,RP6-L-D8B12, D8B12-12-RP9, D112-12-D112, RP9-12-RP9, and RP9-L-RP6(Tables 27-28). Agonist peptides were also identified using FDC-P2 cellproliferation assays (Table 29). Monomer peptides with IGF-1R agonistactivity included G33-lig, G33, S175, D815, lig-D815, RP31-IGF, and D815(Table 29). Dimer peptides with IGF-1R agonist activity includedRP6-RP9, G33-6-G33, and D815-RP9 (Table 29).

In addition, peptides with agonist or antagonist activity in MCF-7 orMiaPaCa cell proliferation assays were shown to compete against IGF-1for binding to IGF-1R (FIGS. 71A-71F and FIGS. 72A-72E). Potencies ofpeptide competition were determined using the AlphaScreen assay formatfor peptide monomers RP60, RP48, sG33, L-RP9ex, and 12-RP9ex (FIGS.71A-71F). Potencies were also determined for dimer peptidesrRP30-IGF-12-D112, rRP30-IGF-12-RP31-IGF, rRP31-IGF-12-RP30-IGF,rD112-12-RP30-IGF, and rD112-12-D112 (FIGS. 72A-72E).

The biological response of the monomers and dimers in the FDC-P2(myeloid cells; IGF-1R/IGF-1R receptor), MCF-7 (breast cancer cells;hybrid IGF-1R/IR receptor) and MiaPaCa (pancreatic cancer cells; hybridIGF-1R/IR receptor) assays were compared (Table 30). In some instances,a modulatory effect (agonism or antagonism) was seen in certain celllines but not in others. For example, the RP30-IGF peptide exhibitedantagonist activity in FDC-P2 and MiaPaCa cells, but not in MCF-7 cells(Table 30). The C1 peptide exhibited antagonist activity in FDC-P2 andMCF-7 cells, but not in MiaPaCa cells. The RP9-RP6, L-RP9ex, andD8B12-12-RP9 peptides exhibited either antagonist or agonist activitydepending on the cell line used (Table 30). Therefore, it is possible touse the peptides of the invention to target specific cell types withspecific modulatory effects.

TABLE 27 IGF-1R Agonists in MCF-7 Cells Site Cellular Binding PeptideForm. IGF-1R EC₅₀ (M) IC₅₀ (M) Sequence G33 1 1 3.0E−07 6.0E−07

12-RP9ex 1 1 1.0E−05 4.5E−07

L-RP9ex 1 1 4.2E−06 2.2E−06

RP48 Misc. 3.2E−06 6.6E−07

RP60 Misc. 2.1E−06 1.6E−05

RP9-12-RP9 1-1 1-1 2.4E−06 N/A

RP9-L-RP6 1-2 1-1 1.3E−06 N/A

D112-12-RP30-IGF 1-2 1-1 2.5E−07 1.7E−07

D112-12-D112 1-1 1-1 3.5E−07 6.2E−07

RP30-IGF-12-D112 2-1 1-1 4.1E−07 1.1E−06

RP6-L-D8B12 2-6 1-2 7.1E−07 N/A

RP30-IGF-12-RP31-IGF 2-6 1-2 1.1E−07 1.1E−06

RP31-IGF-12-RP30-IGF 6-2 2-1 3.0E−06 7.2E−08

D8B12-12-RP9 6-1 2-1 3.5E−07 N/A

Monomer and dimer peptides which stimulate cell proliferation using theMCF-7 cells. Form. = formula; N/A = not available; Misc. = miscellaneoussequence; Cysteine pairs are shaded and underlined; FY, WF, and Lresidues from Formula 1 and Formula 2 motifs are shaded and shown inbold.

TABLE 28 IGF-1R Agonists in MiaPaCa Cells Site Cellular Binding PeptideForm. IGF-1R EC₅₀ (M) IC₅₀ (M) Sequence RP48 Misc. 7.0E−06 6.6E−07

RP60 Misc. 3.1E−06 1.6E−05

RP9-L-RP6 1-2 1-1 1.0E−06 N/A

D112-12-RP30-IGF 1-2 1-1 2.2E−06 1.7E−07

D112-12-D112 1-1 1-1 2.5E−06 6.2E−07

RP9-12-RP9 1-1 1-1 2.7E−06 N/A

RP6-L-D8B12 2-6 1-2 5.0E−06 N/A

RP31-IGF-12-RP30-IGF 6-2 2-1 4.4E−06 7.2E−08

RP30-IGF-12-RP31-IGF 2-6 1-2 6.8E−05 1.1E−06

D8B12-12-RP9 6-1 2-1 1.0E−06 N/A

Monomer and dimer peptides which stimulate cell proliferation using theMiaPaCa cells. Form. = formula; N/A = not available; Misc. =miscellaneous sequence; Cysteine pairs are shaded and underlined; FY,WF, and L residues from Formula 1 and Formula 2 motifs are shaded andshown in bold.

TABLE 29 IGF-1R Agonists in FDC-P2 Cells Site Cellular Binding PeptideForm. IGF-1R EC₅₀ (M) IC₅₀ (M) Sequence G33-lig 1 1 3.0E−06 7.1E−07

G33 1 1 2.0E−06 6.0E−07

S175 1 1 1.0E−05 7.1E−06

D815 6 2 3.0E−06 1.1E−05

lig-D815 6 2 1.0E−05 2.2E−06

RP31-IGF 6 2 2.0E−05 8.4E−07

D815 6 2 1.0E−06 1.5E−06

RP6-RP9 2-1 1-1 6.0E−06 N/A

G33-6-G33 1-1 1-1 3.0E−06 1.4E−07

D815-RP9 6-1 2-1 3.0E−06 1.1E−06

Monomer and dimer peptides which stimulate cell proliferation using theFDC-P2 cells. Form. = formula; N/A = not available; Lig =Diaminopropionic acid with a 2-aminohydroxyacetyl group (CO—CH2—O—NH2)on the side chain amino group; Cysteine pairs are shaded and underlined;FY, WF, and L residues from Formula 1 and Formula 2 motifs are shadedand shown in bold.

TABLE 30 FDC-P2 FDC-P2 MCF-7 MCF-7 MiaPaCa MiaPaCa Peptide SiteIGFR/Formula Agonist Antagonist Agonist Antagonist Agonist AntagonistMonomers: D815 2 (cys.) 6 + RP30-IGF 1 (cys.) 2 + No No + RP31-IGF 2(cys.) 6 + + No No G33 1 (cys.) 1 + + + RP9 1 1 + No No No No RP6 1(cys.) 2 + + No No C1 1 (cys.) 1 + + No No RP33-IGF 1 (cys.) 2 + + +H2C-A-H6 1 1 ND ND + + RP43 1 (cys.) 2 + No No + RP48 Misc. (cys.) NDND + + RP52 Misc. (cys.) ND ND No No + RP54 Misc. (cys.) ND ND + + RP56Misc. ND ND No No + RP60 Misc. (cys.) ND ND + + Dimers: RP9-12-RP9 1-11-1 ND ND + + D8B12-12-RP9 2-1 (cys.) 2-1 ND ND + + RP9-L-RP6 1-1 (cys.)1-2 + + + RP6-D8B12 1-2 (cys.) 2-2 ND ND + + RP30-IGF-C1 1-1 (cys.) 2-1ND ND + + C1-RP30-IGF 1-1 (cys.) 1-1 ND ND + + RP30-IGF-12-RP30-IGF 1-1(cys.) 2-2 ND ND + + RP30-IGF-12-RP31-IGF 1-2 (cys.) 2-2 ND ND + +RP31-IGF-12-RP30-IGF 2-1 (cys.) 2-2 ND ND + + RP6-D8B12 1-2 (cys.) 2-2ND ND + + ND = Not Done; + = Effect observed; No = No effect observed;Cys. = contains putative cysteine loop.

Incorporated herein by reference in its entirety is the Sequence Listingfor the application, comprising SEQ ID NO:1 to SEQ ID NO:2227. TheSequence Listing is disclosed on three CD-ROMs, designated “CRF”, “Copy1”, and “Copy 2”. The Sequence Listing is a computer-readable ASCII filenamed “18784056PC.app.txt”, created on Sep. 23, 2002, in IBM-PC machineformat, on a MS-Windows®98 operating system. The 18784056PC.app.txt fileis 927,477 bytes in size.

As various changes can be made in the above compositions and methodswithout departing from the scope and spirit of the invention, it isintended that all subject matter contained in the above description,shown in the accompanying drawings, or defined in the appended claims beinterpreted as illustrative, and not in a limiting sense.

The contents of all patents, patent applications, published articles,books, reference manuals, texts and abstracts cited herein are herebyincorporated by reference in their entirety to more fully describe thestate of the art to which the present invention pertains.

1. A method of modulating insulin-like growth factor receptor activityin insulin-like growth factor-responsive mammalian cells comprising:contacting the cells with an amino acid sequence in an amount sufficientto modulate the activity of insulin-like growth factor receptor, whereini) the amino acid sequence comprises a Formula 1 sequence, X₁X₂X₃X₄X₅,such that X₁, X₂, and X₅ are selected from the group consisting ofphenylalanine and tyrosine, X₃ is selected from the group consisting ofaspartic acid, glutamic acid, glycine and serine, and X₄ is selectedfrom group consisting of tryptophan, tyrosine and phenylalanine; and ii)with the proviso that the amino acid sequence is not insulin,insulin-like growth factor, an anti-insulin receptor antibody, ananti-insulin-like growth factor receptor antibody, or fragments thereof.